Isolation, identification, and uses of antifungal compounds

ABSTRACT

In one aspect, the invention relates to isolated compounds useful as antifungal agents, for example, compounds having a structure represented by a formula: wherein R 1  is hydrogen or hydroxyl; wherein R 2  is hydrogen, a-xylose or β-xylose; and wherein R 3  and R 4  are each hydrogen or together oxygen, or a pharmaceutically acceptable salt thereof; methods of isolating and purifying same; pharmaceutical compositions comprising same; agricultural compositions comprising same; and methods of treating and/or preventing fungal infections using same. In one aspect, R 2  is not hydrogen or β-xylose when R 1  is hydroxyl and R 3  and R 4  are together oxygen. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of InternationalApplication No. PCT/US2012/071089, filed Dec. 20, 2012, which claimspriority to U.S. Provisional Application No. 61/675,802, filed Jul. 25,2012, and U.S. application Ser. No. 13/332,345, filed Dec. 20, 2011,each of which are incorporated herein fully by reference.

BACKGROUND

Fungal pathogens remain a growing source of infections in plants,animals, and humans. For example, fungal infections of agriculturalplants continue to impact crop growth and yield. Without effectivetreatments, agricultural crop yields can suffer or be destroyed. Longutilized synthetic agents have now become ineffective due increasingincidence of resistance in pathogens. While the contributions ofsynthetic pesticides and herbicides have proven to be of utmost value,problems of toxicity and slow degradability of these compounds in theenvironment is a continuing problem. To this end, alternative methods offungal pathogen control are needed.

In mammals, incidence of superficial or cutaneous fungal infection isnot uncommon. However of greater concern, the incidence of systemicfungal infections has risen significantly. Systemic fungal infectionsare associated with increased morbidity and mortality, especially inimmunocomprimised patients. The widespread use of antifungals in mammalshas conferred increasing resistance in many pathogens.

As such, novel and alternative methods of treating fungal infections areneeded. The disclosed compounds, compositions, and methods address theseneeds and other needs.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates tocompounds useful as antifungal agents, methods of isolating andpurifying same, pharmaceutical compositions comprising same,agricultural compositions comprising same, and methods of treatingand/or preventing fungal infections using same.

Disclosed are isolated compounds having a structure represented by aformula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, or apharmaceutically acceptable salt thereof.

Also disclosed are isolated compounds having a structure represented bya formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof.

Also disclosed are pharmaceutical compositions comprising a compound, ora pharmaceutically acceptable salt thereof, having a structurerepresented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, and apharmaceutically acceptable carrier.

Also disclosed are pharmaceutical compositions comprising a compound, ora pharmaceutically acceptable salt thereof, having a structurerepresented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof.

Also disclosed are methods for preparing an antifungal compositioncomprising combining a compound, or a pharmaceutically acceptable saltthereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, with acarrier.

Also disclosed are methods for preparing an antifungal compositioncomprising combining a compound, or a pharmaceutically acceptable saltthereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof.

Also disclosed are methods for the manufacture of a medicament fortreatment or prevention of fungal infection in a subject, comprisingcombining a compound, or a pharmaceutically acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, with apharmaceutically acceptable carrier.

Also disclosed are methods for the manufacture of a medicament fortreatment or prevention of fungal infection in a subject, comprisingcombining a compound, or a pharmaceutically acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof.

Also disclosed are methods for the manufacture of composition fortreatment or prevention of fungal infection in crops, comprisingcombining a compound, or a agriculturally acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, with anagriculturally acceptable carrier.

Also disclosed are methods for the manufacture of composition fortreatment or prevention of fungal infection in crops, comprisingcombining a compound, or a agriculturally acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or 3-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof.

Also disclosed are methods for the treatment or prevention of fungalinfection in a subject, comprising administering to the subject aneffective amount of a compound, or a pharmaceutically acceptable saltthereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, therebytreating or preventing of fungal infection in the subject.

Also disclosed are methods for the treatment or prevention of fungalinfection in a subject, comprising administering to the subject aneffective amount of a compound, or a pharmaceutically acceptable saltthereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof, thereby treating or preventing of fungal infection in thesubject

Also disclosed are methods for the treatment or prevention of fungalinfection in a plant, comprising administering to the plant an effectiveamount of a compound, or a pharmaceutically acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen, therebytreating or preventing of fungal infection in the plant.

Also disclosed are methods for the treatment or prevention of fungalinfection in a plant, comprising administering to the plant an effectiveamount of a compound, or a pharmaceutically acceptable salt thereof,having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen, or a pharmaceutically acceptable saltthereof, thereby treating or preventing of fungal infection in theplant.

Also disclosed are methods for inhibiting fungal growth on a surface,comprising applying to the surface an effective amount of a compound, ora salt thereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen.

Also disclosed are methods for inhibiting fungal growth on a surface,comprising applying to the surface an effective amount of a compound, ora salt thereof, having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen, α-xylose orβ-xylose; and wherein R³ and R⁴ are each hydrogen or are togetheroxygen, wherein R² is not hydrogen or β-xylose when R¹ is hydroxyl andR³ and R⁴ are together oxygen.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1A and FIG. 1B show the Ion Trap MS (ITMS) spectrum of 2: Bk-1097.

FIG. 2 shows the ¹H NMR spectrum in DMSO-d₆. A: Bk-1229, B: Bk-1097.

FIG. 3 shows the COSY spectrum in DMSO-d₆. A: Bk-1229 full spectrum, B:Bk-1229 expanded region, and C: Bk-1097 full spectrum. Numbers show COSYcorrelations between amide and 6-protons.

FIG. 4 shows a graph the TOCSY spectrum in DMSO-d₆. A: Bk-1229 fullspectrum, B: Bk-1229 expanded region, and C: Bk-1097 full spectrum.

FIG. 5 shows a graph that shows the HSQC spectrum in DMSO-d₆. A: Bk-1229full spectrum, B: Bk-1229 expanded region, and C: Bk-1097 full spectrum.

FIG. 6 shows a graph that shows the ROESY spectrum in DMSO-d₆. A:Bk-1229 full spectrum, B: Bk-1229 expanded (fingerprint) region, theassignment of crosspeaks in FIGS. 6(B), and C: Bk-1097 full spectrum.

FIG. 7 shows the HMBC spectrum in DMSO-d₆. A: Bk-1229, and B: Bk-1097.

FIG. 8 shows the ¹³C spectrum of 2 in DMSO-d₆, and the DEPT spectrum of2 in DMSO-d₆. Numbers show the order of carbons as in Table 1.

FIG. 9 shows the structure of fatty acyl amino acid (FAA) of 2 showingCOSY, HMBC and ROESY correlations.

FIG. 10 shows representative structure of Bk-1229.

FIG. 11 shows the structure of fatty acyl amino acid (FAA) of Bk-1229.

FIG. 12 shows HPLC analysis of FDAA derivatives of burkholdines. HPLCchromatograms of FDAA derivatives of: (line a) burkholdines after acidhydrolysis, (line b) DL-beta hydroxy Asp, (line c) DL-Ser, (line d)DL-Asp, showing (1) L-β-hydroxyAsp (9.5 min), (2) L-Asp (10 min), (3)D-β-hydroxyAsp (10.8 min), (4) D-Asp and L-Ser (13.4 min), (5) D-Ser(16.6 min).

FIG. 13 shows HPLC analysis of FDVA derivatives of burkholdines. HPLCchromatograms of FDVA derivatives of: (line a) DL-Asp/DL-Ser mixture,(line b) burkholdines after acid hydrolysis, showing (1) L-Asp (18 min),(2) D-Asp (21.3 min), (3) L-Ser (21.9 min), (4) D-Ser (25.3 min).

FIG. 14: HPLC analysis of dinitrobenzyl (DNB-F) derivative ofburkholdines showing the presence of L-Ser-L-Asn residue, including HPLCchromatogram of dinitrobenzyl derivative of: (line a) L-Ser-D-Asn (22.7min), (line b) L-Ser-L-Asn (20.6 min), (line c) formic acid hydrolysateof burkholdines (20.6 min).

FIG. 15 shows HPLC analysis of FDVA of burkholdines emphasizing thepresence of L-Ser-L-Asn residue. HPLC chromatogram of FDVA derivativeof: (line a) L-Ser-L-Asn (17 min), (line b) L-Ser-D-Asn (19.7 min),(line c) formic acid hydrolysate of burkholdines (17 min), (line d) FDVAonly.

FIG. 16 shows the selected key HMBC, ¹H-¹H COSY and ROESY correlationsfor compounds 3 and 5.

FIG. 17 shows the key ROESY correlations and rotational model for theFAA unit in Bk-1097 (2).

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

A. DEFINITIONS

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “anagent” includes mixtures of two or more such agents, reference to “thecomponent” includes mixtures of two or more such component, and thelike.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed, then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application data are provided in a number of different formats andthat this data represent endpoints and starting points and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point “15” are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15. It is also understood that each unit betweentwo particular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some aspects of the disclosedmethods, the subject has been diagnosed with a need for treatment priorto the administering step. In some aspects of the disclosed methods, thesubject has been identified to be in need of treatment for a disorder,which refers to selection of a subject based upon need for treatment ofthe disorder. It is contemplated that the identification can, in oneaspect, be performed by a person different from the person making thediagnosis. It is also contemplated, in a further aspect, that theadministration can be performed by one who subsequently performed theadministration.

As used herein, the term “plant” can be, but is not limited to, a cereal(wheat, barley, rye, oat, rice, maize, sorghum and related species);beet (sugar beet and fodder beet); pomes, drupes and soft fruit (apples,pears, plums, peaches, almonds, cherries, strawberries, raspberries andblackberries); leguminous plants (beans, lentils, peas, soybeans); oilplants (rape, mustard, poppy, olives, sunflowers, coconut, castor oilplants, cocoa beans, groundnuts); cucumber plants (pumpkins, cucumbers,melons); fiber plants (cotton, flax, hemp, jute); citrus fruit (oranges,lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus,cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae(avocado, cinnamon, camphor) or plants such as tobacco, nuts, coffee,eggplants, sugarcane, tea, pepper, vines including grape-bearing vines,hops, bananas, turf and natural rubber plants, as well as ornamentals(flowers, shrubs, broad-leafed trees, evergreens, geraniums, azaleas,roses, tulips, petunias, orchids, carnations, poinsettias,chrysanthemums; and conifers such as pine, yew, spruce).

As used herein, the terms “Bk-1229” and “burkholdine 1229” refer to anisolated compound having a structure represented by a formula:

wherein R¹ is hydroxyl; wherein R² is β-xylose; and wherein R³ and R⁴are together oxygen. Bk-1229 is referred to as compound 1 in the Exampletitled “Burkholdines from burkholderia ambifaria” described herein.

As used herein, the terms “Bk-1097” or “burkholdine 1097” refer to anisolated compound having a structure represented by a formula:

wherein R¹ is hydroxyl; wherein R² is hydrogen; and wherein R³ and R⁴are together oxygen. Bk-1097 is referred to as compound 2 in the Exampletitled “Burkholdines from burkholderia ambifaria” described herein.

As used herein, the terms “Bk-1215” and “burkholdine 1215” refer to anisolated compound having a structure represented by a formula:

wherein R¹ is hydroxyl; wherein R² is α-xylose; and wherein R³ and R⁴are hydrogen. Bk-1215 is referred to as compound 3 in the Example titled“Burkholdines from burkholderia ambifaria” described herein.

As used herein, the terms “Bk-1119” and “burkholdine 1119” refer to anisolated compound having a structure represented by a formula:

wherein R¹ is hydrogen; wherein R² is β-xylose; and wherein R³ and R⁴are hydrogen. Bk-1119 is referred to as compound 4 in the Example titled“Burkholdines from burkholderia ambifaria” described herein.

As used herein, the terms “Bk-1213” or “burkholdine 1213” refer to anisolated compound having a structure represented by a formula:

wherein R¹ is hydrogen; wherein R² is β-xylose; and wherein R³ and R⁴are together oxygen. Bk-1213 is referred to as compound 5 in the Exampletitled “Burkholdines from burkholderia ambifaria” described herein.

As used herein, the term “inhibiting” refers to the adverse effectsagainst a microorganism, such as retarding, suppressing or stoppinggrowth of the microorganism or killing or lysing the microorganism. Theadverse effect on growth may be temporary or permanent.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with a fungal infection” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by a compoundor composition that anti-fungal or fungicidal activity. As a furtherexample, “diagnosed with a need for anti-fungal treatment” refers tohaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition characterized bya fungal infection. Such a diagnosis can be in reference to a disorder,such as a neurodegenerative disease, and the like, as discussed herein.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a fungal infection)based upon an earlier diagnosis by a person of skill and thereaftersubjected to treatment for the disorder. It is contemplated that theidentification can, in one aspect, be performed by a person differentfrom the person making the diagnosis. It is also contemplated, in afurther aspect, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, sublingual administration, buccaladministration, transdermal administration, administration byinhalation, nasal administration, topical administration, intravaginaladministration, ophthalmic administration, intraaural administration,intracerebral administration, rectal administration, and parenteraladministration, including injectable such as intravenous administration,intra-arterial administration, intramuscular administration, andsubcutaneous administration. Administration can be continuous orintermittent. In various aspects, a preparation can be administeredtherapeutically; that is, administered to treat an existing disease orcondition. In further various aspects, a preparation can be administeredprophylactically; that is, administered for prevention of a disease orcondition.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired result or to have an effect on anundesired condition. For example, a “therapeutically effective amount”refers to an amount that is sufficient to achieve the desiredtherapeutic result or to have an effect on undesired symptoms, but isgenerally insufficient to cause adverse side affects. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

As used herein, “kit” means a collection of at least two componentsconstituting the kit. Together, the components constitute a functionalunit for a given purpose. Individual member components may be physicallypackaged together or separately. For example, a kit comprising aninstruction for using the kit may or may not physically include theinstruction with other individual member components. Instead, theinstruction can be supplied as a separate member component, either in apaper form or an electronic form which may be supplied on computerreadable memory device or downloaded from an internet website, or asrecorded presentation.

As used herein, “instruction(s)” means documents describing relevantmaterials or methodologies pertaining to a kit. These materials mayinclude any combination of the following: background information, listof components and their availability information (purchase information,etc.), brief or detailed protocols for using the kit, trouble-shooting,references, technical support, and any other related documents.Instructions can be supplied with the kit or as a separate membercomponent, either as a paper form or an electronic form which may besupplied on computer readable memory device or downloaded from aninternet website, or as recorded presentation. Instructions can compriseone or multiple documents, and are meant to include future updates.

As used herein, the terms “therapeutic agent” include any synthetic ornaturally occurring biologically active compound or composition ofmatter which, when administered to an organism (human or nonhumananimal), induces a desired pharmacologic, immunogenic, and/orphysiologic effect by local and/or systemic action. The term thereforeencompasses those compounds or chemicals traditionally regarded asdrugs, vaccines, and biopharmaceuticals including molecules such asproteins, peptides, hormones, nucleic acids, gene constructs and thelike. Examples of therapeutic agents are described in well-knownliterature references such as the Merck Index (14th edition), thePhysicians' Desk Reference (64th edition), and The Pharmacological Basisof Therapeutics (12th edition), and they include, without limitation,medicaments; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of a disease orillness; substances that affect the structure or function of the body,or pro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. For example, the term“therapeutic agent” includes compounds or compositions for use in all ofthe major therapeutic areas including, but not limited to, adjuvants;anti-infectives such as antibiotics and antiviral agents; analgesics andanalgesic combinations, anorexics, anti-inflammatory agents,anti-epileptics, local and general anesthetics, hypnotics, sedatives,antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics,antagonists, neuron blocking agents, anticholinergic and cholinomimeticagents, antimuscarinic and muscarinic agents, antiadrenergics,antiarrhythmics, antihypertensive agents, hormones, and nutrients,antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines,antinauseants, antineoplastics, antipruritics, antipyretics;antispasmodics, cardiovascular preparations (including calcium channelblockers, beta-blockers, beta-agonists and antiarrythmics),antihypertensives, diuretics, vasodilators; central nervous systemstimulants; cough and cold preparations; decongestants; diagnostics;hormones; bone growth stimulants and bone resorption inhibitors;immunosuppressives; muscle relaxants; psychostimulants; sedatives;tranquilizers; proteins, peptides, and fragments thereof (whethernaturally occurring, chemically synthesized or recombinantly produced);and nucleic acid molecules (polymeric forms of two or more nucleotides,either ribonucleotides (RNA) or deoxyribonucleotides (DNA) includingboth double- and single-stranded molecules, gene constructs, expressionvectors, antisense molecules and the like), small molecules (e.g.,doxorubicin) and other biologically active macromolecules such as, forexample, proteins and enzymes. The agent may be a biologically activeagent used in medical, including veterinary, applications and inagriculture, such as with plants, as well as other areas. The termtherapeutic agent also includes without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness; orsubstances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

As used herein, “IC₅₀,” is intended to refer to the concentration of asubstance (e.g., a compound or a drug) that is required for 50%inhibition of a biological process, or component of a process, includinga fungus, protein, subunit, etc. For example, IC₅₀ refers to the halfmaximal (50%) inhibitory concentration (IC) of a substance as determinedin a suitable assay.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

The term “agriculturally acceptable” describes a material that is notundesirable for use with crops, i.e., without causing an unacceptablelevel of undesirable effects or interacting in a deleterious manner.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

The term “cyclic group” is used herein to refer to either aryl groups,non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl groups), or both. Cyclic groups have one or more ringsystems that can be substituted or unsubstituted. A cyclic group cancontain one or more aryl groups, one or more non-aryl groups, or one ormore aryl groups and one or more non-aryl groups.

The term “hydroxyl” as used herein is represented by the formula —OH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture.

Compounds described herein can contain one or more asymmetric centersand, thus, potentially give rise to diastereomers and optical isomers.Unless stated to the contrary, the present invention includes all suchpossible diastereomers as well as their racemic mixtures, theirsubstantially pure resolved enantiomers, all possible geometric isomers,and pharmaceutically acceptable salts thereof. Mixtures ofstereoisomers, as well as isolated specific stereoisomers, are alsoincluded. During the course of the synthetic procedures used to preparesuch compounds, or in using racemization or epimerization proceduresknown to those skilled in the art, the products of such procedures canbe a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

B. COMPOUNDS

In one aspect, the invention relates to an isolated compound having astructure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen.

In another aspect, the invention relates to an isolated compound havinga structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; R² is hydrogen, α-xylose orβ-xylose; and R³ and R⁴ are each hydrogen or are together oxygen,wherein R² is not hydrogen or R-xylose when R¹ is hydroxyl and R³ and R⁴are together oxygen, or a pharmaceutically acceptable salt thereof

In one aspect, R³ and R⁴ are each hydrogen. In a further aspect, R¹ ishydrogen. In a still further aspect, R¹ is hydroxyl. In yet a furtheraspect, R² is hydrogen. In yet a further aspect R² has a structurerepresented by a formula:

In yet a further aspect, R² has a structure represented by a formula:

In one aspect, R³ and R⁴ are together oxygen. In a further aspect, R¹ ishydrogen. In a still further aspect, R¹ is hydroxyl. In a yet furtheraspect, R² is hydrogen. In yet a further aspect, R² has a structurerepresented by a formula:

In yet a further aspect, R² has a structure represented by a formula:

In yet another aspect, R¹ is hydroxyl; R² is α-xylose; and R³ and R⁴ areeach hydrogen.

In yet another aspect, R¹ is hydrogen; R² is β-xylose; and R³ and R⁴ areeach hydrogen.

In yet another aspect, R¹ is hydrogen; R² is (-xylose; and R³ and R⁴ aretogether oxygen.

In one aspect, the invention relates to an isolated compound having astructure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is hydrogen or xylose;and wherein R³ and R⁴ are each hydrogen or together oxygen. In oneaspect, R² is not hydrogen or β-xylose when R¹ is hydroxyl and R³ and R⁴are together oxygen. In a further aspect, the compound is present in atleast about 50% purity. In a still further aspect, the compound ispresent in at least about 75% purity. In a yet further aspect, thecompound is present in at least about 90% purity. In a yet furtheraspect, the compound is present in at least about 95% purity. In yet afurther aspect, the compound is present in at least about 99% purity.

In one aspect, the compound is isolated from beta-proteobacteria. In afurther aspect, the compound is isolated from Burkholderia orPseudomonas species. In a still further aspect, the compound is isolatedfrom Burkholderia species. In a yet further aspect, the compound isisolated from Burkholderia ambifaria. In yet a further aspect, thecompound is isolated from Burkholderia ambifaria strain 2.2 N.

C. ANTI-FUNGAL ACTIVITY

The utility of the compounds in accordance with the present invention asinhibitors of fungal cell growth, can be demonstrated by methodologyknown in the art.

In one aspect, the compound exhibits antifungal activity againstBotrytis, Mycosphaerella, Cercospora, Rhizoctonia, Monilinia,Phytophthora, Altemaria, Candida, Saccharomyces, Aspergillus,Pseudocercosporella, Cladosporium, Chaetomium, Fusarium, Colletotrichum,Epidermophyton, Trichophyton, or Microsporum.

In one aspect, the compound is cytotoxic. In a further aspect, thecompound inhibits fungal cell growth compound with a MIC of less thanabout 12.5 μg/ml. In a still further aspect, the compound inhibitsfungal cell growth compound with a MIC of less than about 1.6 μg/ml. Ina yet further aspect, the compound inhibits fungal cell growth compoundwith a MIC of less than about 0.4 μg/ml.

In one aspect, the compound exhibits of about at least 50% fungal cellgrowth inhibition with an IC of less than about 2000 ppm. In a furtheraspect, the compound exhibits of about at least 50% fungal cell growthinhibition with an IC of less than about 1000 ppm. In a still furtheraspect, the compound exhibits of about at least 50% fungal cell growthinhibition with an IC of less than about 500 ppm. In a yet furtheraspect, the compound exhibits of about at least 50% fungal cell growthinhibition with an IC of less than about 100 ppm. In yet a furtheraspect, the compound exhibits of about at least 50% fungal cell growthinhibition with an IC of less than about 20 ppm.

In one aspect, the compound exhibits of about at least 75% fungal cellgrowth inhibition with an IC of less than about 2000 ppm. In a furtheraspect, the compound exhibits of about at least 75% fungal cell growthinhibition with an IC of less than about 1000 ppm. In a still furtheraspect, the compound exhibits of about at least 75% fungal cell growthinhibition with an IC of less than about 500 ppm. In a yet furtheraspect, the compound exhibits of about at least 75% fungal cell growthinhibition with an IC of less than about 100 ppm. In yet a furtheraspect, the compound exhibits of about at least 75% fungal cell growthinhibition with an IC of less than about 20 ppm.

In one aspect, the compound exhibits of about at least 90% fungal cellgrowth inhibition with an IC of less than about 2000 ppm. In a furtheraspect, the compound exhibits of about at least 90% fungal cell growthinhibition with an IC of less than about 1000 ppm. In a still furtheraspect, the compound exhibits of about at least 90% fungal cell growthinhibition with an IC of less than about 500 ppm. In a yet furtheraspect, the compound exhibits of about at least 90% fungal cell growthinhibition with an IC of less than about 100 ppm. In yet a furtheraspect, the compound exhibits of about at least 90% fungal cell growthinhibition with an IC of less than about 20 ppm.

D. METHODS OF PROVIDING THE COMPOUNDS

In one aspect, the invention relates to methods of producing thedisclosed compounds useful as fungal growth inhibitors, which can beuseful in the treatment or prevention of fungal infections and otherconditions in which inhibition or killing of fungal cells is desired.

The compounds of this invention can be prepared by employing cellculturing, extraction, and isolation methods as shown in the followingsection, in addition to other standard manipulations that are known inthe literature, exemplified in the experimental sections or clear to oneskilled in the art.

Methods used to isolate the compounds of this invention are prepared byemploying methods as shown in the following section, in addition toother standard isolation methods known in the literature or to oneskilled in the art. The following examples are provided so that theinvention might be more fully understood, are illustrative only, andshould not be construed as limiting.

In a one aspect, a compound comprises the product of the disclosedmethods. In a still further aspect, the invention comprises apharmaceutical composition comprising a therapeutically effective amountof the product of the disclosed methods and a pharmaceuticallyacceptable carrier. In a further aspect, the invention comprises aagricultural composition comprising a therapeutically effective amountof the product of the disclosed methods and an agriculturally acceptablecarrier. In a still further aspect, the invention comprises a method formanufacturing a medicament comprising combining at least one compound ofany of disclosed compounds or at least one product of the disclosedmethods with a pharmaceutically acceptable carrier or diluent.

In one aspect, the invention relates to a method of isolating thedisclosed compounds comprising the steps of providing a fermentationbroth of bacterial cultures of Burkholderia ambifaria and extracting thecompound from the fermentation broth. In a further aspect, the methodfurther comprises spray-drying or freeze-drying the fermentation broth.In a still further aspect, the method further comprises purifying thecompound by solvent-liquid extraction. In a still further aspect, themethod further comprises purifying the compound by solid-liquidextraction. In a still further aspect, the method further comprisespurifying the compound by revered phase high pressure liquidchromatography.

In one aspect, the method further comprises heat-treating thefermentation broth to a temperature sufficient to kill the bacteria. Ina further aspect, the method further comprises comprising heating thefermentation broth to at least about 80° C.

In one aspect, the compound is isolated from beta-proteobacteria. In afurther aspect, the compound is isolated from Burkholderia orPseudomonas species. In a still further aspect, the compound is isolatedfrom Burkholderia species. In a yet further aspect, the compound isisolated from Burkholderia ambifaria. In yet a further aspect, thecompound is isolated from Burkholderia ambifaria strain 2.2 N.

It is contemplated that each disclosed methods can further compriseadditional steps, manipulations, and/or components. It is alsocontemplated that any one or more step, manipulation, and/or componentcan be optionally omitted from the invention. It is understood that adisclosed methods can be used to provide the disclosed compounds. It isalso understood that the products of the disclosed methods can beemployed in the disclosed methods of using.

E. PHARMACEUTICAL COMPOSITIONS

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compounds. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of at least one disclosed compound or at least one product of adisclosed method and a pharmaceutically acceptable carrier.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, vaginal, topical,and parenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein. In a preferredaspect, the compositions disclosed herein can be administeredintraperitoneally. For example, during intraabdominal surgery (e.g.,resection of an abcess), the disclosed compositions can be used as awash. In this way, cells that were not excised and/or cells that may beunattached but still present in the body, can be killed uponreadhereing.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The disclosed compositions can be in solution, suspension (for example,incorporated into microparticles, liposomes, or cells). These can betargeted to a particular cell type via antibodies, receptors, orreceptor ligands.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical or vaginal use such as, for example, an aerosol,cream, ointment, lotion, dusting powder, mouth washes, gargles, and thelike. Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal or vaginal administration wherein the carrier is a solid. Inone aspect, the mixture forms unit dose suppositories. Suitable carriersinclude cocoa butter and other materials commonly used in the art. Thesuppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

Thus, the compositions can comprise, in addition to the disclosedcompositions or for example, lipid or liposomal formulations, such aswith cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. A lipid or liposomal formulation can further compriseproteins to facilitate cellular penetration, or targeting a particularcell, if desired. Administration of a composition comprising a compoundand a cationic liposome can be administered to the blood afferent to atarget organ or inhaled into the respiratory tract to target cells ofthe respiratory tract. Regarding liposomes, see, e.g., Brigham et al.Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et al Proc. Natl.Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore,the compound can be administered as a component of a microcapsule wherethe diffusion of the compound or delivery of the compound from themicrocapsule is designed for a specific rate or dosage.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

F. AGRICULTURAL COMPOSITIONS

In one aspect, the invention relates to agricultural compositionscomprising the disclosed compounds. That is, a agricultural compositioncan be provided comprising a therapeutically effective amount of atleast one disclosed compound or at least one product of a disclosedmethod and a agriculturally acceptable carrier.

In certain aspects, the disclosed agricultural compositions comprise thedisclosed compounds (including agriculturally acceptable salt(s)thereof) as an active ingredient, a agriculturally acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for foliar or systemicadministration.

The instant compounds may sprayed, atomized, dusted, scattered, coated,or poured on the subject, although the most suitable route in any givencase will depend on the particular host, and nature and severity of theconditions for which the active ingredient is being administered. Theagricultural compositions can be conveniently presented as an emulsion,paste, solution, powder, dust, granulate, polymeric encapsulation orcombination and prepared by any of the methods well known in the art ofagrichemicals.

As used herein, the term “agricultually acceptable salts” refers tosalts prepared from acceptable bases or acids. When the compound of thepresent invention is acidic, its corresponding salt can be convenientlyprepared from agricultually acceptable bases, including inorganic basesand organic bases. Salts derived from such inorganic bases includealuminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous,lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zincand the like salts. Particularly preferred are the ammonium, calcium,magnesium, potassium and sodium salts. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary, and tertiary amines, as well as cyclic amines and substitutedamines such as naturally occurring and synthesized substituted amines.Other agricultually acceptable organic bases from which salts can beformed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “agricultually acceptable acids”, includesinorganic acids, organic acids, and salts prepared therefrom, forexample, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or agricultually acceptablesalts thereof, of this invention can be combined as the activeingredient in intimate admixture with a agricultually carrier accordingto conventional agrochemical manufacturing techniques. The carrier cantake a wide variety of forms depending on the form of preparationdesired for administration, e.g., spraying or dusting. Thus, theagricultural compositions of the present invention can be presented as apowder for administration such as dusting. Further, the compositions canbe presented as granules, as a solution, as a suspension in an aqueousliquid, as a non-aqueous liquid, as an oil-in-water emulsion or as awater-in-oil liquid emulsion. In addition to the common dosage forms setout above, the compounds of the invention, and/or agricultuallyacceptable salt(s) thereof, can also be administered by controlledrelease means and/or delivery devices. The compositions can be preparedby any of the methods of known in the art. In general, such methodsinclude a step of bringing into association the active ingredient withthe carrier that constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both. The product can then be conveniently shaped intothe desired presentation.

Thus, the agricultural compositions of this invention can include aagricultually acceptable carrier and a compound or a agricultuallyacceptable salt of the compounds of the invention. The compounds of theinvention, or agricultually acceptable salts thereof, can also beincluded in agricultural compositions in combination with one or moreother therapeutically active compounds.

The agricultural carrier employed can be, for example, a solid or aliquid. Examples of such carriers include mineral substances, solvents,dispersants, wetting agents, tackifiers, thickeners, binders, orfertilizers. In preparing the compositions for administration, anyconvenient agricultural media can be employed. For example, thecompositions may also contain further adjuvants such as stabilizers,antifoams, viscosity regulators, binders or tackifiers as well asfertilizers, micronutrient donors or other formulations for obtainingspecial effects. Optionally, whereas it is preferred to formulatecommercial products as concentrates, whereas the end user will normallyuse dilute formulations.

The disclosed agricultural compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above the treatment or prevention of fungal infectionfrom Altemaria, Aspergillus, Botrytis, Cercospora, Cercosporidium,Erysiphe, Geotrichum, Mycosphaerella, Mucor, Phoma, Phytophthora,Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus,Septoria, Sporothrix, Stemphylium, Trichophyton, and Verticillium.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

G. METHODS OF USING THE COMPOSITION

1. Treatment Methods

a. Pharmaceutical Treatments

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety ofconditions and/or diseases associated with fungal infection.

Fungal infections can manifest as superficial, cutaneous, or systemicinfections. Examples of superficial or cutaneous classes of fungalinfections include: dermatophytosis, dermatomycosis, onychomycosis,piedra, or candidiasis. Specific examples of superficial or cutaneousfungal infections include, but are not limited to: tinea capitis, tineafavosa, tinea corporis, tinea faciei, tinea pedis, tinea manuum, tineaimbricata, tinea cruris, tinea barbae, tinea nigra, tinea ungium, oralcandidiasis, candidal vaginitis, candidal intertrigo, perianalcandidiasis, candidal paronychia, erosio interdigitalis blastomycetica,chronic mucocutaneous candidiasis, and seborrhoeic dermatitis. Examplesof systemic fungal infections include: aspergillosis, blastomycosis,invasive candidiasis, coccidioidomycosis, cryptococcosis,histoplasmosis, paracoccidioidomycosis, zygomycosis, and pneumocystosis.Typical fungal pathogen species infections may include: Aspergillus,Candida, Cryptococcus, Histoplasma, Pneumocystis, Saccharomyces,Epidermophyton, Microsporum, Trichophyton, Scytalidium, Pitvrosporum,Coccidioides, Histoplasma, Paracoccidioides, Blastomyces, Sporothrix,Candida, Trichosporon, Pneumocystis, Exophiala, Fonsecaea, Geotrichum,Pseudallescheria, and Rhizopus.

Thus, in some aspects of the disclosed method, the fungal infection istinea capitis, tinea favosa, tinea corporis, tinea faciei, tinea pedis,tinea manuum, tinea imbricata, tinea cruris, tinea barbae, tinea nigra,tinea ungium, oral candidiasis, candidal vaginitis, candidal intertrigo,perianal candidiasis, candidal paronychia, erosio interdigitalisblastomycetica, chronic mucocutaneous candidiasis, and seborrhoeicdermatitis. In a further aspect, the fungal infection is aspergillosis,blastomycosis, invasive candidiasis, coccidioidomycosis, cryptococcosis,histoplasmosis, paracoccidioidomycosis, zygomycosis, and pneumocystosis.

In one aspect, the invention relates to a method for the treatment of afungal infection in a subject comprising the step of administering tothe subject at least one disclosed compound or at least one disclosedproduct in a dosage and amount effective to treat the disorder in thesubject. In a further aspect, the subject is a mammal, for example, ahuman. In a further aspect, the mammal has been diagnosed with a needfor treatment of the disorder prior to the administering step. In afurther aspect, the method further comprises the step of identifying asubject in need of treatment of the disorder. In a still further aspect,the fungal infection is cutaneous or superficial. In yet a furtheraspect, the fungal infection is systemic.

In one aspect, the method further comprises treating dermatophytosis,dermatomycosis, onychomycosis, piedra, or candidiasis. In furtheraspect, the treatment method further comprises treating tinea capitis,tinea favosa, tinea corporis, tinea faciei, tinea pedis, tinea manuum,tinea imbricata, tinea cruris, tinea barbae, tinea nigra, tinea ungium,oral candidiasis, candidal vaginitis, candidal intertrigo, perianalcandidiasis, candidal paronychia, erosio interdigitalis blastomycetica,chronic mucocutaneous candidiasis, or seborrhoeic dermatitis. In a yetfurther aspect, the method further comprises treating aspergillosis,blastomycosis, invasive candidiasis, coccidioidomycosis, cryptococcosis,histoplasmosis, paracoccidioidomycosis, zygomycosis, or pneumocystosis.

In one aspect, the method further comprising the step of identifying thesubject as having a need for treatment or prevention of fungalinfection. In a further aspect, the subject (e.g., mammal) isimmunocompromised or immunosuppressed.

In one aspect, the method further comprising treating a fungal infectionfrom Aspergillus, Candida, Cryptococcus, Histoplasma, Pneumocystis,Saccharomyces or a combination thereof. In a further aspect, the fungalinfection is from Epidermophyton, Microsporum, Trichophyton,Scytalidium, Pityrosporum, Coccidioides, Histoplasma, Paracoccidioides,Blastomyces, Sporothrix, Candida, Cryptococcus, Trichosporon,Pneumocystis, Exophiala, Fonsecaea, Geotrichum, Pseudallescheria, orRhizopus.

In one aspect, the method further comprises the route of administrationis oral, buccal, sublingual, nasal, topical, rectal, vaginal, parenteralor a combination thereof. In a further aspect, the route ofadministration is intramuscular, intravenous, intraocular,intraperitoneal, subcutaneous, or combination thereof.

In one aspect, the disclosed compositions can be administered by I.V.,by injection and/or an I.V. drip.

For therapeutic uses, pharmaceutical compositions and formulations cancontain an effective amount of active for treating the disorder. Thespecific effective amount for any particular subject will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the identity and activity of the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration; the route of administration; therate of excretion of the specific composition employed; the duration ofthe treatment; drugs used in combination or coincidental with thespecific composition employed and like factors well known in the medicalarts. For example, it is well within the skill of the art to start dosesof a composition at levels lower than those required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved. One can also evaluate the particularaspects of the medical history, signs, symptoms, and objectivelaboratory tests that are known to be useful in evaluating the status ofa subject in need of attention for the treatment of ischemia-reperfusioninjury, trauma, drug/toxicant induced injury, neurodegenerative disease,cancer, or other diseases and/or conditions. These signs, symptoms, andobjective laboratory tests will vary, depending upon the particulardisease or condition being treated or prevented, as will be known to anyclinician who treats such patients or a researcher conductingexperimentation in this field. For example, if, based on a comparisonwith an appropriate control group and/or knowledge of the normalprogression of the disease in the general population or the particularindividual: 1) a subject's physical condition is shown to be improved,2) the progression of the disease or condition is shown to bestabilized, or slowed, or reversed, or 3) the need for other medicationsfor treating the disease or condition is lessened or obviated, then aparticular treatment regimen will be considered efficacious. If desired,the effective daily dose can be divided into multiple doses for purposesof administration. Consequently, single dose compositions can containsuch amounts or submultiples thereof to make up the daily dose.

An effective amount of the composition can also be determined bypreparing a series of compositions comprising varying amounts of thedisclosed compounds and determining the release characteristics in vivoand in vitro and matching these characteristics with specificpharmaceutical delivery needs, inter alia, subject body weight, diseasecondition and the like.

The dosage for the compositions can be adjusted by the individualphysician or the subject in the event of any counterindications. Dosagecan vary, and can be administered in one or more dose administrationsdaily, for one or several days. Guidance can be found in the literaturefor appropriate dosages for given classes of pharmaceutical products.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder is affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage can be 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315,320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455,460, 465, 470, 475, 480, 485, 490, 495, or 500 mg/kg, where any of thestated values can be upper of lower end points of a range.

Example dosages are disclosed herein. For example, when treating asubject with a fungal infection, or when preventing a fungal infectionin a subject, the disclosed compounds or a pharmaceutically acceptablesalt or hydrate thereof can be administered at a dosage of from 1 toabout 500 mg/kg of the subject, can be administered at a dosage of from10 to about 200 mg/kg of the subject, can be administered at a dosage offrom 10 to about 100 mg/kg of the subject, or can be administered at adosage of from 20 to about 500 mg/kg of the subject.

The compositions can be administered alone or combination with otherantimicrobrial drugs. Combination therapy can present advantages oversingle-agent therapies: lower treatment failure rate, lowercase-fatality ratios, slower development of resistance and consequently,less money needed for the development of new drugs. Antimicrobrial drugsinclude. The disclosed compounds can also be administered in combinationwith surgery. For example, the disclosed compounds can be administeredprior to, during or after surgery. Administration during surgery can beas a bathing solution for the operation site. The resected part can alsobe bathed in the disclosed compounds.

It is contemplated that the disclosed compounds can be adminsteredbefore, simultaneously, or after the administration of one or moreadditional antimicrobial agents. While not wishing to be bound bytheory, it is believed that the disclosed compounds, in combination withone or more chemotherapeutic drugs, can have an augmented or synergisticeffect on the subject. Further, the disclosed compounds, in combinationwith one or more antimicrobial drugs, can be individually given indosages lower than the one or more antimicrobial drugs would betypically adminstered as single-agent therapies.

The disclosed compositions can also be employed to prevent fungalinfection in a subject, such as in immunocomprimised patients. In oneaspect, such a method comprises administering to the subject aprophylactically effective amount of or a pharmaceutically acceptablesalt or hydrate thereof. It is understood that the dosage and/orfrequency needed to prevent (i.e. maintenance dose) may be less (e.g.,once vs. twice daily) of the dosage (e.g., half) needed to effecttreatment of a fungal infections. Thus, in maintenance, a suitabledosage of the disclosed compounds or a pharmaceutically acceptable saltor hydrate thereof can be from 0.5 to about 250 mg/kg of the subject,can be administered at a dosage of from 5 to about 100 mg/kg of thesubject, can be administered at a dosage of from 5 to about 50 mg/kg ofthe subject, or can be administered at a dosage of from 10 to about 250mg/kg of the subject.

b. Agricultural Treatment

The compounds disclosed herein are useful for treating, preventing,ameliorating, controlling or reducing the risk of a variety ofconditions and/or diseases associated with fungal infection.

Fungal infections can be various fungal microorganisms including, butnot limited to: Botrytis, Mycosphaerella, Cercospora, Rhizoctonia,Monilinia, Phytophthora, Alternaria, Aspergillus, Pseudocercosporella,Cladosporium, Chaetomium, Fusarium, or Colletotrichum.

In one aspect, the invention relates to a method for the treatment orprevention of fungal infection in a plant, comprising administering tothe plant an effective amount of the disclosed compound. In a furtheraspect, the method further comprises the step of identifying the plantas having a need for treatment or prevention of fungal infection.

In one aspect, the invention relates to a method for the treatment orprevention of fungal infection in a plant, comprising administering tothe plant an effective amount of the disclosed compound where the fungalmicroorganism is from Alternaria, Aspergillus, Botrytis, Cercospora,Cercosporidium, Erysiphe, Geotrichum, Mycosphaerella, Mucor, Phoma,Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia,Rhizopus, Septoria, Sporothrix, Stemphylium, Trichophyton, andVerticillium.

In one aspect, the method further comprises an amount of the disclosedcompounds that is therapeutically effective. In a further aspect, themethod comprises an amount that is prophylactically effective.

In one aspect, the invention relates to a method for the treatment orprevention of fungal infection in a plant, comprising administering tothe plant an effective amount of the disclosed compound where thecompound is application is foliar. In a further aspect, the method ofapplication is systemic. In a still further aspect, the method ofapplication is to the soil. In yet a further aspect, the method ofapplication is to the seeds.

In one aspect, the invention relates to a method for the treatment orprevention of fungal infection in a plant, further comprisingadministration the disclosed compounds by spraying, atomizing, dusting,scattering, coating, or pouring.

In one aspect, the invention relates to a method for the treatment orprevention of fungal infection in a plant, comprising administering tothe plant an effective amount of the disclosed compound where the plantis corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco,cotton, flax, safflower, oats, and canola; fruits and vegetables such astomato, pepper, cucumber, lettuce, green beans, lima beans, peas,cantaloupe, musk melon, citrus fruits, grapes, and banana; andornamentals and cut flowers such as geraniums, azaleas, roses, tulips,petunias, orchids, carnations, poinsettias, chrysanthemums; and coniferssuch as pine, yew, spruce.

For therapeutic uses, agricultural compositions and formulations cancontain an effective amount of active ingredient for treating thedisorder. The specific effective amount for any particular subject willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the identity and activity of thespecific composition employed; the the time of administration; the routeof administration; the duration of the treatment; agents used incombination or coincidental with the specific composition employed andlike factors well known in the agricultural arts.

An effective amount of the composition can also be determined bypreparing a series of compositions comprising varying amounts of thedisclosed compounds and determining the release characteristics in vivoand in vitro and matching these characteristics with specific deliveryneeds, disease conditions and the like.

As used herein, an “effective” dose or regime of treatment with thedisclosed composition against a particular fungal pathogen is one whoseapplication achieves at least 20%, in a further aspect at least 50%, ina still further aspect at least 80%, and a yet further aspect at least90%, disease control against that pathogen. An effective dose or regimemay be achieved by adjusting the level of active ingredient(s) in thecomposition and/or the amount or frequency of compositions is applied.

The rates of application are can be about 5 g to about 2 kg of activeingredient (a.i.) per hectare (ha). In a further aspect, the rate isabout from 10 g to about 1 kg a.i./ha. In a still further aspect, therate is about from 20 gm to about 600 gm a.i./ha. Ranges for theadministration of the compositions are those large enough to produce thedesired effect in which the symptoms of the disorder is affected. Thedosage should not be so large as to cause adverse side effects, such astoxicity or death.

c. Environmental Treatment

In one aspect, the invention relates to a method for the inhibitingfungal growth on a surface comprising the step of applying to thesurface at least one disclosed compound or at least one disclosedproduct in a dosage and amount effective to inhibit fungal growth. In afurther aspect, the method further comprises the step of identifyingfungus on the surface prior to application. In a further aspect, thecompound is cytoxic to the fungus.

In one aspect, the method further comprises application to wood, paper,plastic, metal, stone, natural and synthetic composite, natural andsynthetic fabric, or leather. In a further aspect, the surface isheating, ventilation, and air-conditioning (HVAC) surfaces, insulation,wall-board, kitchen surfaces, bathroom surfaces, or other surface foundin a building. In a still further aspect, the surface is found in acommercial or residential building.

2. Manufacture of a Medicament

The present invention is further directed to a method for themanufacture of a medicament for treating fungal infections (e.g.,treatment of one or more skin and/or systemic infections associated withfungal pathogens) in subjects (e.g., mammals, e.g., humans) comprisingcombining one or more disclosed compounds, products, or compositionswith a pharmaceutically acceptable carrier or diluent. Thus, in oneaspect, the invention relates to a method for manufacturing a medicamentcomprising combining at least one disclosed compound or at least onedisclosed product with a pharmaceutically acceptable carrier or diluent.

3. Kits

In one aspect, the invention relates to a kit comprising a disclosedcompound or a product of a disclosed method and one or more of at leastone agent known to inhibit fungal growth; at least one agent known toincrease fungal growth; at least one agent known to treat a fungalinfection; at least one agent known to treat a disease of uncontrolledfungal cell proliferation; or instructions for treating a disorderassociated with fungal infections. In a further aspect, the at least onecompound or the at least one product and the at least one agent areco-formulated. In a further aspect, the at least one compound or the atleast one product and the at least one agent are co-packaged.

In a further aspect, the kit comprises a disclosed compound or a productof a disclosed method.

In a further aspect, the at least one compound and the at least oneagent are co-formulated. In a still further aspect, the at least onecompound and the at least one agent are co-packaged.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound and/orproduct and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connectionwith the disclosed methods of making, the disclosed methods of using,and/or the disclosed compositions.

4. Non-Medical Uses

Also provided are the uses of the disclosed compounds and products aspharmacological tools in the development and standardization of in vitroand in vivo test systems for the evaluation of the antifungal activityor, as part of the search for new antifungal agents. In a furtheraspect, the invention relates to the use of a disclosed compound or adisclosed product as pharmacological tools in the development andstandardization of in vitro and in vivo test systems for the evaluationof antifungal activity as part of the search for new therapeutic agentsor treatment protocols.

H. COMPOSITIONS WITH SIMILAR FUNCTIONS

It is understood that the compositions disclosed herein have certainfunctions, such as antifungal activities or anti-proliferativeactivities. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures willultimately achieve the same result, for example, inhibition of fungalcell proliferation.

I. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Several methods for preparing the compounds of this invention areillustrated in the following Examples. Starting materials and therequisite intermediates are in some cases are commercially available, orcan be prepared according to literature procedures or as illustratedherein.

The following exemplary compounds of the invention were synthesized. TheExamples are provided herein to illustrate the invention, and should notbe construed as limiting the invention in any way.

1. General Methods

Cultures obtained from B. ambifaria 2.2N strain were isolated fromspray-dried cells, for example, by sequential solvent extraction withdichloromethane, 2-propanol and 2-propanol: H₂O. Optionally, thefractions may be further purified, for example on an open C₁₈ column.Optionally, the compounds can be further purified, for example byreverse phase HPLC. Further purification of the compounds resulted inessentially pure product.

2. Fermentation of Burkholderia

Compounds of the present invention can be produced by culturing thepreviously discussed microorganism in an an aqueous nutrient mediumcontaining sources of carbon and nitrogen. Sources of carbon in thenutrient medium can include carbohydrates such as sucrose, glucose,glycerol, and the like. The exact quantity and number of carbonsource(s) will vary, and will depend on, including but not limited to,other constituents in the medium, desired growth rate, and the like.Sources of nitrogen can include amino acids such as glycine, proline,methionine, and the like. An example of the process for production ofthe compounds of the present invention comprises inoculating themicroorganism into a suitable culture medium, and then culturing underaerobic conditions. In one example, the disclosed compounds may beisolated from the aerobic fermentation of a culture of Burkholderiaambifaria.

(1) Example 1

Initial growth studies of the 2.2 N strain were completed in 2 differentmedia (¼ strength Tryptic Soy Broth (TSB)+0.2% Sucrose and strength TSB)and specific growth rate and doubling time were determined. Workingstock culture was prepared in strength TSB+0.2% Sucrose media and usedas inocula for future experiments. Initial shake flask experiments werecompleted using three production media to evaluate anti-fungal yield andvariability. The production media tested were: strength TSB+0.2%Sucrose; % strength TSB+0.2% Glucose; and KB20 media with glycerol.Anti-fungal material, with complete inhibition at 20 ppm in petri-dishstudy against Botrytis was isolated using ¼ strength TSB+0.2% Sucrosemedia. This media was selected for further studies in shake flaskexperiments.

Process scale-up evaluations were carried in 7.5 L fermentors with 5 Lworking volume. Various process conditions were tested to determineoptimal production environment, including media (¼ strength TSB+0.2%Sucrose; ¼ strength TSB+0.2% Glucose; and KB20 media), pH (6 & 7),agitation (120 and 250 rpm), dissolved oxygen set at 20% (cascaded toagitation). All the fermentation batches were heat-killed beforespray-drying or freeze-drying. Different temperatures (range from50-121° C.) were tested to study the heat-stability of the activeingredient and establish optimal conditions to heat-kill the bacteriawhile maintaining 100% anti-fungal activity. Heat treatment at 80° C.for 10 min resulted in 100% anti-fungal activity. Increase intemperature to 100° C. resulted in substantial loss of activity.

Relatively high load of variant forms in the culture streaked from thefermentor was noticed. The original 2.2N strain was purified andseparated amber colored and mucoid variant by repeated streaking on agarplates. The purified strain do not show any improvement in theanti-fungal activity compared to original strain. However, mucoidvariant form resulted in very low to no activity.

The fermentation broth was initially spray-dried at an Inlet temperatureof 150-160° C. and lower outlet temperature of 60-65° C. compared to anoutlet temperature of 85-100° C. Different batches were then tested forfreeze-drying (to preserve the heat-labile antifungal activity) todetermine whether continuous exposure to higher temperature duringspray-drying resulted in lower activity. Spray-drying and freeze-dryingmethod for product recovery do not show any significant difference inactivity. The heat-killed fermentation broth was concentrated using aRotary evaporator and spray-dried to produce concentrated material. Theheat-treated controls and concentrated product yielded from the Rota-vapwere very active with complete inhibition of Botrytis at 20 ppm.Additionally, untreated fermentation culture broth resulted in higherproduct yield. Filter-sterilized culture supernatant resulted in verylow to no activity. Cell breakage using sonicator did not show anyincrease in activity, which was similar to the untreated control.Sonication for a longer period of time resulted in heat generation andfurther degradation of the active ingredient, indicating scale-up ofsonication is not a feasible technology.

(2) Example 2

Working stock cultures of strain 2.2 N were first prepared in an aqueousnutrient medium consisting of 1.5% (w/v) Tryptic Soy Broth (TSB), 0.2%(w/v) sucrose, 0.25% (w/v) K₂HPO₄, and 0.5% (w/v) NH₄Cl. Cultures (250L) were produced in 300 L working volume fermentors. The inocula werestaged from 100 mL to 25 L in the same medium in 10-fold increasingincrements. Cultures were incubated for 32 h at 30° C. with aerationmaintained at 30% dissolved oxygen, and pH 6 was maintained by additionof 17% (v/v) NH₄OH. Cells were recovered by centrifugation (5000×g) in acontinuous flow centrifuge and cell pastes packaged in plastic bags andfrozen. Cell pastes were defrosted, adjusted to 30% moisture content byaddition of water, and spray dried at an inlet temperature of 170° C.and outlet temperature of 100° C. The dried product moisture content was5%.

3. Extraction and Isolation of Compounds

A. Example 1

A slurry of spray dried cells (35 g) in dichloromethane (DCM, 400 mL)was stirred at room temperature for 20 h. The slurry was filteredthrough 3 cm bed of celite and washed with DCM (200 mL). The solidresidue was stirred with 2-propanol (350 mL) for 1 h. The solvent wasremoved by vacuum filtration and the residue was stirred with2-propanol:H₂O (30:70, 400 mL) for 12 h at room temperature. Theresulting suspension was transferred to twelve 50 mL-Falcon tubes andcentrifuged for 1 h at 4000 rpm. The supernatant was discarded and thepellets were combined and dissolved in acetonitrile:water (1:1) (100mL). Lyophilization of this suspension gave 5.3 g crude extract. 1 gcrude extract was suspended in methanol:DCM (80:20, 25 mL), thencentrifuged for 1 h at 4000 rpm. The supernatant was discarded and theremaining solid was suspended in 20 mL H₂O and lyophilized to give 250mg extract enriched with burkholdines (presently disclosed compounds).The bacterial extract (250 mg) was suspended in a volume of 1.5 mL waterwith 2% acetonitrile and applied to the top of an open C₁₈ column. Thecolumn was eluted with a gradient of acetonitrile-water (2%, 10%, 25%,50% and 100%) and five fractions were collected (F1-F5). According toLC/MS, F4 (8.5 mg) was found to be highly enriched in a mixture ofburkholdines. F4 was further purified by reversed phase high pressureliquid chromatography (RP-HPLC) using preparative Atlantis-T3 column (10mm×250 mm). Compounds were eluted with a linear gradient from 95%solvent A (0.07% trifluoroacetic acid in water) to 60% solvent B (0.07%trifluoroacetic acid in acetonitrile) over 95 min at 5 mL/min. Theeluent was monitored at 205 nm and peaks at 59 min (Bk-1229) werecollected and lyophilized to yield Bk-1229 (400 μg) as off-white powder.

Bk-1229 (1): UV (CH₃CN:H₂O 1:1) λ_(max) nm (log ε) 230 (1.54), 274(1.08); IR (KRS-5 cell, acetonitrile)^(v) _(max) 3274, 2919, 1698, 1683,1670, 1652, 1635, 1558, 1540, 1519, 1507 cm-¹; ¹H and ¹³C NMR, seeSupporting Tables 1 and 2; HRESIMS [M+H]⁺ m/z 1230.5746 (calcd forC₅₂H₈₄N₁₁O₂₃, 1230.5735, 0.3 ppm).

A. Example 2—Isolation of Compound 2

A slurry of spray dried cells (35 g) in dichloromethane (DCM, 400 mL)was stirred at room temperature for 20 h. The slurry was filteredthrough 3 cm bed of celite and washed with DCM (200 mL). The solidresidue was stirred with 2-propanol (350 mL) for 1 h. The solvent wasremoved by vacuum filtration and the residue was stirred with2-propanol:H₂O (30:70, 400 mL) for 12 h at room temperature. Theresulting suspension was transferred to twelve 50 mL-Falcon tubes andcentrifuged for 1 h at 4000 rpm. The supernatant was discarded and thepellets were combined and dissolved in acetonitrile:water (1:1) (100mL). Lyophilization of this suspension gave 5.3 g crude extract. 1 gcrude extract was suspended in methanol:DCM (80:20, 25 mL), thencentrifuged for 1 h at 4000 rpm. The supernatant was discarded and theremaining solid was suspended in 20 mL H₂O and lyophilized to give 250mg extract enriched with burkholdines. The bacterial extract (250 mg)was suspended in a volume of 1.5 mL water with 2% acetonitrile andapplied to the top of an open C₁₈ column. The column was eluted with agradient of acetonitrile-water (2%, 10%, 25%, 50% and 100%) and fivefractions were collected (F1-F5). According to LC/MS, F4 (8.5 mg) wasfound to be highly enriched in a mixture of burkholdines. F4 was furtherpurified by reversed phase high pressure liquid chromatography (RP-HPLC)using preparative Atlantis-T3 column (10 mm×250 mm). Compounds wereeluted with a linear gradient from 95% solvent A (0.07% trifluoroaceticacid in water) to 60% solvent B (0.07% trifluoroacetic acid inacetonitrile) over 95 min at 5 mL/min. The eluent was monitored at 205nm and peaks at 64 min (Bk-1097) were collected and lyophilized to yieldBk-1097 (300 μg) as off-white powder.

Bk1097 (2): UV (CH₃CN:H₂O 1:1) λ_(max) nm (log ε) 232 (1.48), 276(1.09); IR (KRS-5 cell, acetonitrile)^(v) _(max) 3273, 2920, 1669, 1651,1635, 1557, 1539, 1519, 1507 cm-¹; ¹H and ¹³C NMR, see Supporting Tables1 and 3; HRESIMS [M+H]⁺ m/z 1098.5321 (calcd for C₄₇H₇₆N₁₁O₁₉,1098.5313, 0.2 ppm).

TABLE 1 HSQC ASSIGNMENTS 1: Bk-1229 2: Bk-1097 No. δC δH Assignment No.δC δH Assignment 1 13.5 0.84 C18—CH3 FAA 1 13.4 0.84 C18—CH3 FAA 2 21-351.24 (C9-C17)—CH2 2 21-31 1.24 (C9-C17)—CH2 FAA FAA 3 29.6 1.53 C8—CH2FAA 3 32.9 1.35, C8—CH2 FAA 1.40 4 36.2 2.38 β Asn II 4 36.3 2.38 β AsnII 2.42 β Asn I 2.43 β Asn I 2.58 β Asn I 2.58 β Asn I 2.62 β Asn II2.62 β Asn II 5 38.4 1.34 C4—CH2 FAA 5 38.6 1.34 C4—CH2 FAA 1.77 1.75 640.3 2.30 C2—CH2 FAA 6 40.4 2.31 C2—CH2 FAA 2.41 2.41 7 41.6 3.55 α Gly7 41.3 3.56 α Gly 3.84 3.83 8 43.6 4.20 C3—CH FAA 8 43.7 4.19 C3—CH FAA9 49.3 4.56 α Asn I 9 49.3 4.56 α Asn I 10 49.4 4.49 α Asn II 10 49.54.49 α Asn II 11 54.5 4.30 α Ser I 11 54.7 4.30 α Ser I 12 54.7 4.05 αSer II 12 54.9 4.09 α Ser II 13 54.8 4.63 α Hydroxy Asn 13 55.0 4.64 αHydroxy Asn 14 59.9 4.04 α HydroxyTyr 14 60.0 4.05 α HydroxyTyr 15 60.53.46 β Ser II 15 60.6 3.45 β Ser II 16 61.1 3.59 β Ser I 16 61.1 3.58 βSer I 17 65.2 3.04 C5 Xylose 17 67.0 3.43 C5—CH FAA 3.70 C5 18 66.5 3.48C5—CH FAA 18 68.4 3.60 C7—CH FAA 19 68.9 3.33 C4 Xylose 19 70.3 4.97 βHydroxyTyr 20 70.1 4.96 β HydroxyTyr 20 71.4 3.96 β HydroxyAsn 21 71.23.96 β HydroxyAsn 21 75.3 2.92 C6—CH FAA 22 72.5 2.98 C2 Xylose 22 114.16.65 C3, C5 HydroxyTyr 23 73.7 3.06 C6—CH FAA 23 126.5 7.12 C2, C6HydroxyTyr 24 75.9 3.09 C3 Xylose 25 76.4 3.72 C7—CH FAA 26 101.27 4.17C1 Xylose 27 114.1 6.64 C3, C5 HydroxyTyr 28 126.6 7.12 C2, C6HydroxyTyr

TABLE 2 HMBC AND ROESY CORRELATIONS FOR BK-1229 (1) IN DMSO-D6 (600MHz). Assignment δC δH HMBC ROESY β Hydroxy Tyr α 59.9 4.04 β NH C2—H,C6—H β 70.1 4.96 α β-OH C2—H, C6—H β-OH 5.65 α β C2—H, OH C6—H C1 131.7NA C2 and C6 126.6 7.12 β, C2, C4, C6 α β β-OH NH C3—H, C5—H C3 and C5114.1 6.64 C1, C3, C4, C5 OH C2—H, C6—H C4 155.8 OH 9.25 C3, C4, C5 β-OHC3—H, C5—H NH 7.82 β-OH C2—H, NH (Ser II) α (Ser II) α β C6—H C—O 170.6Asn I α 49.3 4.56 β, C—O, NH (Gly) γ-C—O β 36.2 2.42 α, C—O, 2.58 γ-NH22.58 γ-C—O 2.42 γ-C—O 170.4 NA γ-NH2 6.91 β 7.34 7.34 γ-CO—O 6.91 β NH7.73 C—O α β α (β-Hydroxy (Hydroxy Tyr) Tyr) C—O 167.6 NA Gly α 41.63.55 C—O α NH NH (Asn II) 3.84 NH 7.66 α α (Asn I) C—O 169.5 NA Asn II α49.4 4.49 β, C—O, NH NH (Ser I) γ-C—O β 36.2 2.38 α, C—O, 2.62 NH γ-NH22.62 γ-C—O 2.38 γ-C—O 170.6 NA γ-NH2 6.91 β 7.34 β 7.34 γ-C—O 6.91 NH8.34 C—O α β (Ser I) NH (Ser I) (G1y) C—O 167.7 NA Ser I α 54.5 4.30 β,C—O NH β NH (Hydroxy Asn) β 61.1 3.59 C—O NH β NH OH (Hydroxy Asn) OH4.79 α β NH β NH 7.75 α β OH β (Asn II) α (Asn II) C—O 169.4 NAβ-Hydroxy- Asn α 54.8 4.63 C—O NH β NH (FAA) β 71.2 3.96 α, C—O, α α-OHγ-NH2 γ-C—O β-OH 5.62 α, β, γ-C—O β γ-NH2 γ-C—O 172.6 NA γ-NH2 6.8 7.2 ββ—OH 7.2 6.8 NH 7.80 C—O (Ser I) α β β—OH NH (FAA) α (Ser I) β (Ser I)C—O 166.5 NA FAA NH 7.32 C3—CH α NH NH (Hydroxy (Hydroxy (Ser II) Asn)Asn) C1—C—O 170.5 NA C2—CH2 40.3 2.30 C—O C3—CH C4—CH2 NH (Ser II) 2.41C3—CH 43.6 4.20 NH C2—CH2 C4—CH2 C5—OH NH (Ser II) C4—CH2 38.4 1.34 1.77C5—OH C6—CH C3—CH C6—OH 1.77 1.34 C5—CH C5—CH 66.5 3.48 C4—CH2 C5—OH4.06 C4—CH2, C5—CH, C6—CH C6—CH 73.7 3.06 C7—CH C6—OH C4—CH2 C6—OH 4.12C5—CH, C4—CH2 C5—CH C6—CH, C7—CH C7—CH 76.4 3.72 C6—OH C5—OH C5—CHC8—CH2 (C9-C17)—CH2 C1 — CH Xy C8—CH2 29.6 1.53 C7—CH C6—CH C6—OH C2 —OH (C9-C17)—CH2 C1 — CH Xy (C9-C17)—CH2 (21-35) 1.24 C18—CH3 C18—CH3C8—CH2 C6—CH C7—CH C2 — OH Xy C1 — CH Xy C18—CH3 13.5 0.85 (C9-C17)—CH2(C9-C17)—CH2 Ser II α 54.7 4.05 β NH β 60.5 3.46 OH OH 4.90 α, β β NH NH8.09 C—O α OH C2 — CH2 NH (Tyr) NH (FAA) C3 — CH (FAA) (FAA) (FAA) C—O171.4 NA Xylose (Xy) C1—CH 101.2 4.17 C7—CH C7 — CH (FAA) C6 — CH C8 —CH2 C5—CH2 C2—OH Xy (FAA) (FAA) Xy C2—CH 72.5 2.98 C1—CHXy C2—OH XyC2—OH 4.94 C1—CHXy C1—CH Xy C2—CH Xy C3—CH 75.9 3.09 (C2—CH, C3—OH XyC4—CH) Xy C3—OH 4.93 C3—CH Xy C3—CH Xy C4—CH Xy C4—CH 68.9 3.33 C4—OH4.92 (C4—CH, C4—CH Xy C5—CH2) Xy 65.2 3.04 (C1—CH, 3.70 C4—CH CC1—CH XyC8 — CH2 C3—CH, Xy (FAA) C4—CH) Xy 3.70 (C1—CH, 3.04 C4—OH C3—OH XyC3—CH, Xy C4—CH) Xy *Bolded regions show ROESY correlations betweenresidues.

TABLE 3 HMBC AND ROESY CORRELATIONS FOR BK-1097 (2) IN DMSO-D6(600 MHz).Assignment δC δH J (Hz) HMBC ROESY B-Hydroxy Tyr α 60.0 4.05 4.0, 7.6 βC2—H, C6—H β 70.3 4.97 4.0 C2, C6 α C2—H, C6—H β-OH 5.62 4.0 α β C2—H,C6—H C1 131.7 NA C2 and C6 126.5 7.12 β, C2, C4, C6 α β C3—H, β-OH NHC5—H C3 and C5 114.1 6.65 Cl, C3, C4, C2—H, OH C5 C6—H C4 156.1 NA OH9.25 C3, C4, C.5 C3—H, β-OH C5—H NH 7.88 7.6 α α (Ser II) C2—H, (HydroxyC6—H Tyr) C—O 170.4 NA Asn I α 49.3 4.56 3.9, 5.7 β, γ-C—O β NH (Gly) β36.3 2.43 3.9 α, γ-C—O 2.58 2.58 2.43 γ-C—O 171.0 NA γ-NH2 6.92 3.8 β,γ-C—O 7.34 β 7.34 γ-C—O 6.92 NH 7.77 5.7 C—O α (Hydroxy (Hydroxy Tyr)Tyr) C—O 167.6 NA Gly α 41.3 3.56 3.9, 7.8 C—O 3.83 NH 3.83 3.56 NH 7.697.8 α α (Asn I) C—O 169.8 NA Asn II α 49.5 4.49 4.0, 5.9 β, C—O β NH NH(Ser I) β 36.3 2.38 5.9 α, C—O α γ-NH2 2.62 γ-C—O 171.1 NA γ-NH2 6.92 β,γ-C—O 7.34 β 7.34 γ-C—O 6.92 NH 8.34 4.0 α β α (Gly) C—O 167.8 NA Ser Iα 54.7 4.30 3.8, 5.9 β, C—O β NH (Hydroxy Asn) β 61.1 3.58 3.8 α, C—O αOH NH OH 4.81 β β NH 7.76 5.9 β α (Asn II) C—O 169.4 NA β-Hydroxy Asn α55.0 4.64 4.0, 7.7 C—O, β NH γ-C—O (FAA) β 71.4 3.96 4.0 α, C—O, α NH OHγ-C—O β-OH 5.66 4.0 α,β β NH γ-C—O 173.1 NA γ-NH2 6.82 7.20 β β-OH 7.206.82 NH 7.82 7.7 C—O (Ser I) α β OH NH (FAA) C—O 167.5 NA FAA NH 7.3611.4 C—O α (Hydroxy (Hydroxy Asn) Asn) C1—C—O 170.5 NA C2—CH2 40.4 2.314.0, 7.6 C—O C3—CH 2.41 7.6 C3—CH C3—CH 43.7 4.19 4.0, 11.4 C—O C4—CH238.6 1.34 4.0 1.75 4.0, 11.5, 7.7 C5—CH 67.0 3.43 C5—OH C6—OH C4—CH2C3—CH C5—OH 4.40 C5—CH, C6—CH C6—CH 75.3 2.92 3.9, 7.6 C5—CH C6—OH C7—CHC5—OH C4—CH2 C6—OH 4.11 7.6 C5—CH, C6—CH C7—CH 68.4 3.60 3.9, 7.8 C7—OHC8—CH2 C9—CH2 C6—CH C7—OH 4.04 3.9, 7.8 C6—CH, C7—CH, C8—CH2 C8—CH2 32.91.35 3.9 1.40 (C9-C17)—CH2 (21.6- 1.24 C18—CH3 CH2 30.7) (1.24) C18—CH313.4 0.84 3.9 (C9-C17)—CH2 C18—C3 C7—CH C7—OH C6—CH C6—OH Ser II α 54.94.09 3.9, 5.9 β, C—O NH NH (Hydroxy Tyr) β 60.6 3.45 5.9 α OH OH 4.91 αβ NH 8.09 3.9 C—O (FAA) C2 — CH2 (FAA) 170.9 NA *Bolded regions showROESY correlation between residues.

4. Analytical Methods

¹H, ¹H—¹H_(g)COSY, ¹H—¹H dqfCOSY, ¹H—¹H TOCSY, ¹H—¹³C HSQC, ¹H-¹³C HMBC,¹H—¹H ROESY, DEPT and ¹³C were carried out at 25° C. NMR data werecollected using either a 500 MHz Varian INOVA-NMR spectrometer with a 3mm Nalorac MDBG probe or 600 MHz Varian INOVA-NMR spectrometer with acryoprobe with standard pulse sequences. The IR spectrum was recorded ona JASCO FTIR-420 spectrophotometer. UV spectra were recorded on anhp-8552A diode array spectrophotometer. Electrospray ionization massspectrometry (ESI-MS), Liquid chromatography-mass spectrometry (LC/MS),and Fourier transform-ion cyclotron resonance (FT-ICR) were run at theUniversity of Utah Mass Spectrometry and Proteomics Core Facility.

Acid Hydrolysis and FDAA Analysis (Marfey's method) was performed on thepresently disclosed compounds. In a thick walled microvial, Bk-1229 andBk-1097 (100 μg each) were hydrolyzed in 250 μL of 6 N HCl containing0.1% phenol (w/v). The vial was sealed and heated at 110° C. for 20 h.The reaction was allowed to cool and concentrated to dryness bylyophilization. The residue was dissolved in 100 μL water, andsubsequently 300 μL of 1 M aqueous NaHCO3 and 250 μL of 1% (w/v) ofNα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (L-FDAA) in acetone wereadded. This mixture was heated at 50° C. for 1.5 h and then allowed tocool to room temperature. The L-FDAA derivatives were separated byRP-HPLC using a C18 column (4.6 mm×150 mm). A linear gradient of 10%acetonitrile in 0.1 M NH4OAc (pH 5) to 50% acetonitrile in 0.1 M NH4OAcover 60 min was used at 1 mL/min. The eluent was monitored at 340 nm(FIG. 12).

FDVA Derivatization of the Hydrolysate Burkholdines was then performed.The hydrolysate of Bk-1229 and Bk-1097 was obtained as described aboveand derivatized with 1% (w/v) ofNα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) in acetone. Thereaction was heated at 40° C. for 1 h. The L-FDVA derivatives wereseparated by RP-HPLC as described before (FIG. 13).

Comparing the retention times of FDAA derivatives of L and DL standardamino acids and the hydrolysate of burkholdines, L-Hydroxy-Asp (9.5min), L-Asp (10 min), D-Asp (13.4 min), L-Ser (13.4 min), D-Ser (16.6min) and Gly (18.3 min) were detected. D-Asp and L-Ser peaks overlapped.Burkholdines were derivatized with L-FDVA and both D-Asp (21.3 min) andL-Ser (21.9 min) were identified (FIGS. 12 and 13).

Formic Acid Hydrolysis and Analysis. In 5 replicate microcentrifugetubes, freshly prepared 2% formic acid (200 μl) was added to lyophilizedburkholdines (50 μg). The tubes were heated to 108° C. in a dry heatblock, with variable incubation times from 5 min to 1.5 h. Reactionswere monitored by TLC (silica gel; 13:5:2 butanol:water:acetic acid) forpresence of intermediate hydrolysis products. Upon completion the tubeswere frozen and lyophilized to dryness. Standards of D-Asp-L-Ser andL-Asp-L-Ser were synthesized by the University of Utah DNA/Peptide CoreFacility. Lyophilized formic acid hydrolysis products and the standardswere then derivatized with 2,4-dinitro-1-fluorobenzene (DNB-F) and FDVA.For DNB-F derivatization, samples and standards were dissolved in boratebuffer (40 μl; 100 mM at pH 9) and a solution of DNB-F (60 μl; 10 mM inacetonitrile). All mixtures were vortexed and heated at 65° C. for 30min in a dry heat block and then allowed to cool. The DNB-F derivativeswere separated by RP-HPLC using C18 column (4.6 mm×150 mm). A gradientof 5% acetonitrile/water to 80% for 30 min then to 95% for 40 min at 1mL/min was used. The column was kept at 25° C. and the eluent wasmonitored at 230 nm (FIG. 14). The FDVA derivatization method wasperformed as described above.

Sugar Analysis for Bk-1229. Arabinose (3.3 mmol), xylose (3.3 mmol) andBk-1229 (1.2 mmol) were lyophilized in silanized screw cap tubes anddesiccated in vacuo over P₂O₅ for 3 days. 0.5 mL of 10% benzoyl chloridein pyridine (v/v) was added under nitrogen and the tubes were incubatedat 37° C. for 10 h and then left at room temperature for 14 h. Eachsample was diluted with 9 volumes of water and mixed by vortex for 1min. 0-perbenzoylated derivatives were purified on Bond-Elut C₁₈disposable columns (Varian) and washed with water (6 mL), 25%acetonitrile-water (3 mL), 50% acetonitrile-water (3 mL) and 100%acetonitrile (3 mL). 100% acetonitrile fractions were dried and thendissolved in acetonitrile for HPLC analysis. The fractions were appliedto a C₁₈ column (4.6 mm×150 mm) and eluted with acetonitrile-water75%-100% over 20 min at 1 mL/min. The eluent was monitored at 230 nm(FIG. 14).

5. Biological Assays

The present compounds demonstrate potent antifungal activity against abroad sprectrum of fungal species as demonstrated by the followingexamples.

A. Bioassay (In Vitro)

(1) Example 1

Anti-fungal activity for the present compounds was determined usingmicrotitre format. Microtitre plates (96 wells: rows A-H, columns 1-12)were filled by the following protocol to give 2-fold serial dilutions ofeach of Bk-1229 and Bk-1097. Samples of Bk-1229 and Bk-1097 weredissolved in 1/10-strength BHIB containing 0.1% (w/v) dimethylsulfoxide(DMSO). Aliquots (50 μL) of 1/10-strength BHIB containing 0.1 (w/v) %DMSO (pH 7.4) were added to all wells except those in column 1. Thewells in column I received 100 μL of the solutions of Bk-1229 or Bk-1097and 50 μL was removed and mixed with the medium in the second well. Thisprocess was repeated through well 11, at which point an aliquot (50 μL)was removed and discarded. Well 12 was the blank (positive growth)control, BHIB+DMSO only. The test microorganisms employed in thisexample were Saccharomyces cerevisiae, Candida albicans, and Aspergillusniger, which were obtained from the Virginia Tech Microbiology CultureCollection. Cells of S. cerevisiae and C. albicans were grown in sterile1/10-strength Brain Heart Infusion Broth (BHIB) and incubated at 30° C.for 18 h. Cells were counted as colony forming units on 1/10-strengthBHIB agar. Spore suspensions of A. niger were prepared from lawns ofsporulating colonies on 1/10-strength BHIB agar. The number of sporeswas determined by counting spores microscopically in a Petroff-Hausercounting chamber. The resulting culture or spore suspensions werediluted to 10⁵ cells or spores/mL. Following the preparation of thedilution series, 50 μL of microbial inocula (10⁵/mL) were added to eachwell in a row. The concentration of Bk-1229 and Bk-1097 ranged from 50to 0.05 μg/mL. After the plates were incubated at 30° C. for 4 days,measurement of minimal inhibitory concentration (MIC) results were readby comparing the turbidity (due to microbial growth) of each test wellto the positive control wells. The measured MICs for each ofburkholdines are summarized as follow:

TABLE 12 MIC (μg/mL) Compound S. cerevisiae C. albicans A. niger Bk-1229(1) 0.4 12.5 12.5 Bk-1097 (2) 1.6 12.5 1.6 Amphotericin B 25 25 25

(2) Example 2

Contact petri dish studies were completed testing Dominion BioSciencespray dried material against a list of plant diseases. The results areshown in Table 4. The experimental material (VBC80006) was prepared with80% “formulated” heat-killed material Results (lowest rate) for completeinhibition with continuous exposure to spray dried material fromDominion BioScience against Botrytis: 20 ppm, Mycosphaerella: 100 ppm,Cercospora: 500 ppm, Rhizoctonia: 2000 ppm, Sclerotinia: >5000 ppm,Pythium: >5000 ppm, Monilinia: >5000 ppm, Phytophthora>2000 ppm.Botrytis was selected as the fungal pathogen for QC assays. Additionalpetridish assays and on-plant testing confirmed the activity on Botrytisof the produced material. Reduction in growth of Alternaria andColletotrichum on exposure to the produced killed-microbial material wasmeasured as well.

TABLE 4 Small scale in vitro assay results with various Burkholderia labsamples. Numbers are lowest PPM to obtain greater than 90% inhibition ofplant disease spreading in petriplate. (NT = not tested). VBC 80% =VBC80006 (80% fermentation solids of Burkholderia) VBC 80% VBC 80% VBC80% Plant Disease DBS material A.I A.I., A.I, Botrytis cin. 20 20-50 NT400 Mycosphaerella 100 NT NT NT Cercospora 500 NT NT NT Rhizoctonia sol.2000 NT NT >2500 Monilinia >5000 NT NT 1000 Sclerotinia scler. >5000 NTNT >10000 Pythium >5000 NT NT 10000 Phytophthora inf. >2000 NT NT >5000Alternaria alt. NT NT 500 500 Aspergillus ni. NT NT 500 NTPseudocercosporella NT NT 500 NT Cladosporium clad. NT NT 500 NTPenicillium fun. NT NT No effect NT Chaetomium glo. NT NT 500 NTFusarium ox. NT NT 500 >5000 Pyricularia or. NT NT NT NT Erysiphe gram.NT NT NT NT Spaerotheca ful. NT NT NT NT Leptosphaeria nod. NT NT NT NTPlasmopara vit. NT NT NT NT Colletotrichum NT NT NT 500

b. Bioassay (In Vivo)

A lab study on individual grapes was performed to study the effects onBotrytis in more detail. Table 5 through Table 8 show the results ofdifferent application timing of VBC80006 after inoculation withBotrytis, different rates of VBC80006 as well as storage conditions ofthe grapes at either 21° C. or −5° C. In this example, the pathogen andcompound were applied to the subject at the same time. The results showactivity of VBC80006 to reduce decay development as well as the severityof decay (disease index=numeric system). A dose effect was visible whengrapes were stored at 21° C. and the test scored at 5 days (end point).Three applications of VBC80006 at the higher dose appears to improveefficacy (Table 6). In Table 8 the results are shown of higher ratesagainst a mixture of Botrytis and Penicillium.

TABLE 5 Laboratory study of control of Botrytis decay in table grapes.VBC80006 and Botrytis suspensions were applied at the same time. * 3 =lesion > 3 mm with superficial fungal growth; 2 = lesion > 3 mm withoutsuperficial fungal growth; 1 = lesion < 3 mm with brown discolorationaround pin-hole; 0 = no lesion or brown discoloration. Treatment, rateExamination parameter applied and incubation % % temperature and timeDisease Lesion Decay Incidence in days index area 0 + 1 group* 2 + 3group* VBC80006, 500 PPM, 76.2 18.4 21.5 78.5 21° C., 5 D VBC80006, 5000PPM, 41.3 7.3 64.5 35.5 21° C., 5 D UTC 92.0 50.8 8.0 92.0Non-inoculated 0.7 0.0 100.0 0.0 VBC80006, 500 PPM, 55.3 18.2 43.0 57.0−5° C., 21 D VBC80006, 5000 PPM, 42.3 12.9 59.0 41.0 −5° C., 21 D UTC85.3 45.2 3.0 97.0 Non-inoculated 16.0 2.2 81.0 19.0

TABLE 6 Laboratory study of control of Botrytis decay in table grapes.VBC80006 suspension was applied at different times after Botrytisinoculation. Grapes were held for 5 days at 21° C. Examination parameter% % Treatment, rate, and Disease Lesion Decay Incidence treatmentschedule index area 0 + 1 group* 2 + 3 group* VBC80006, 500 PPM, 76.021.0 28.0 72.0 immediately after inoculation VBC80006, 500 PPM, 83.323.0 10.0 90.0  3 hrs after inoculation VBC80006, 500 PPM, 82.0 17.014.0 86.0 24 hrs after inoculation VBC80006, 500 PPM, 63.3 12.7 34.066.0 immediate + 24 hrs + 48 hrs after inoculation VBC80006, 5000 PPM,44.7 11.1 54.0 46.0 immediately after inoculation VBC80006, 5000 PPM,32.7 7.4 72.0 28.0  3 hrs after inoculation VBC80006, 5000 PPM, 50.7 8.056.0 44.0 24 hrs after inoculation VBC80006, 5000 PPM, 37.3 2.9 76.024.0 immediately + 24 hrs + 48 hrs after inoculation UTC 92.0 50.8 8.092.0 Non-inoculated 0.7 0 100.0 0

TABLE 7 Laboratory study of control of Botrytis decay in table grapes.VBC80006 suspension was applied at different times after Botrytisinoculation. Grapes were held for 21 days at −5 ° C. Examinationparameter % % Treatment, rate, and Disease Lesion Decay Incidencetreatment schedule index area 0 + 1 group* 2 + 3 group* VBC80006, 50058.0 20.3 40.0 60.0 PPM, immediately after inoculation VBC80006, 50052.7 16.2 46.0 54.0 PPM, 3 hrs after inoculation VBC80006, 5000 48.714.6 52.0 48.0 PPM, immediately after inoculation VBC80006, 5000 36.011.1 66.0 34.0 PPM, 3 hrs after inoculation UTC 84.0 45.1 4.0 96.0Non-inoculated 15.3 2.3 82.0 18.0

TABLE 8 Laboratory study of control of both Botrytis and Penicilliumdecay in table grapes. VBC80006 and Botrytis suspensions were applied atthe same time. Grapes were held for 5 days at 21 ° C. Examinationparameter % % Disease Lesion Decay Incidence Treatment, rate appliedindex area 0 + 1 group* 2 + 3 group* VBC80006, 2500 PPM 36.2 3.7 71.128.9 VBC80006, 5000 PPM 28.5 1.9 78.9 21.1 Fungicide reference 18.2 0.396.7 3.3 UTC 91.1 26.8 8.9 91.1

In one example, banana leaves were innoculated with black sigatokadisease (Mvcosphaerella), Table 9 and 10 show inhibition of sporegermination and germ tube growth of the disease.

TABLE 9 In vivo study on banana leaves with black Sigatoka(Mycosphaerella fijiensis). Germ tube inhibition as percentage of check.VBC80006 is 80% fermentation solids of Burkholderia. % Germ tubeinhibition Treatment 0.01 PPM 0.1 PPM 1.0 PPM 10.0 PPM 100.0 PPM EC50*VBC80006 23.5 9.0 16.0 85.0 100.0 3.0 PPM *EC50 = concentration in PPMrequired for 50% inhibition.

TABLE 10 In vivo study on banana leaves with black Sigatoka(Mycosphaerella fijiensis). Inhibition of spore germination. VBC80006 is80% fermentation solids of Burkholderia. % of non-germinating sporesTreatment Check 0.01 PPM 0.1 PPM 1.0 PPM 10.0 PPM 100.0 PPM VBC80006 017 0 0 50.0 100.0

c. Field Evaluations

In one example, Burkholderia killed-microbial material (VBC80006) wastested on banana trees to evaluate the antifungal activity against blacksikatoga disease. The produced Burkholderia killed-microbial material(VBC80006) sprayed in an oil-in-water emulsion reduced the incidence ofblack Sigatoka especially on the lower part of banana leaves as good asDiThane over a 46 day exposure to natural infestation. (See Table 11).

TABLE 11 In vivo study on banana leaves with black Sigatoka(Mycosphaerella fijiensis). Inhibition of disease severity by VBC80006.Applications made with airbrush at equivalent of 23 liters per ha sprayvolume, and using as spreader sticker Spraytex oil at 7 liter/ha plus NP7 at 1% the rate of the oil.One application, treatments replicated 6X inRCB design. Natural levels of disease pressure. Disease Severity (daysafter application) Treatment Side of leaf 25 days 40 days 46 daysVBC80006, 100 PPM Upper surface 6 28 41 VBC80006, 1000 PPM Upper surface3 31 61 VBC80006, 2500 PPM Upper surface 0 34 68 DiThane Upper surface 036 74 UTC Upper surface 0 68 96 VBC80006, 100 PPM Lower 0 6 27 surfaceVBC80006, 1000 PPM Lower 0 7 8 surface VBC80006, 2500 PPM Lower 0 4 7surface DiThane Lower 0 4 9 surface UTC Lower 0 49 84 surface

Limited field tests of the Burkholderia killed-microbial material So farlimited field tests of the Burkholderia killed-microbial material didnot show any efficacy in the reduction of the plant diseases such asPythium. Greenhouse and on-leaf assays showed promise in a few cases.On-plant assays done confirmed activity of VBC80006 on Botrytis at 200PPM. Other tests showed moderate activity against Rhizoctonia: 5000 ppm,Sclerotinia: 5000 ppm, Phytophthora 5000 ppm, Alternaria: 5000 ppm,Fusarium: 1000 ppm, Pyricularia: 1000 ppm, Erysiphe: 5000 ppm,Spaerotheca: 5000 ppm, Leptosphaeria: 1000 ppm, Plasmopara: 5000 ppm.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otheraspects of the invention will be apparent to those skilled in the artfrom consideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

6. Burkholdines from Burkholderia Ambifaria

As described above, burkholdines are cyclic lipopeptides with unusualantifungal potency, making them promising leads as a new class ofantifungal agents. A recent report using knockout mutagenesis (Thomsonand Denis 2012) indicates that these and related compounds, such asoccidiofungins, xylocandins, and cepacidines, can also be synonymouswith the long-known hemolytic virulence factors found in diverseBurkholderia isolates. Because of their possible roles in causing(Burkholderia infections) or curing (fungal infections) human infectiousdiseases, it is important to fully define their structures and theirbiological activities using pure compounds. The structures of threefurther burkholdines, Bk-1119, Bk-1213, and Bk-1215, were analyzed andare described which were elucidated using spectroscopic methods. Theabsolute configuration of this compound class was determined for thefirst time using a combination of spectroscopy and chemical degradationtechniques. Antifungal and hemolytic activities were assessed for fivepure burkholdines, representative of the structural diversity of thislipopeptide class. All of the burkholdines were potent antifungal andhemolytic agents, validating their probable role in virulence. However,one of the burkholdines (Bk-1119) exhibit>30-fold selectivity for fungiversus sheep erythrocytes and was more than 25-fold more potent thanamphotericin against some fungal strains. Therefore, burkholdines,including the described burkholdines, have potential to selectivelytarget fungal infections.

There remains a critical need to develop new antifungal agents for humanhealth and agricultural purposes.^(1,2) In an effort to target fungalinfections, the bacterium Burkholderia ambifaria 2.2N was isolated froma soil sample collected at a parking lot adjacent to the buildinghousing the Microbiology Department at the Pennsylvania State Universityand shown to possess potent antifungal activity.³ The technology waslicensed by Dominion Biosciences, Inc. and fermentation optimization wasperformed at the Penn State Bioprocessing Facility in 1997-1998.Following discovery that the antifungal activity was heat-resistant, thecell pastes produced at the Bioprocessing Facility were spray dried andshown to be active. Those spray-dried powders were used in extensivefield trials performed throughout the United States in 1998,demonstrating broad-spectrum anti-fungal activity,³⁻⁵ but the majoractive components were long uncharacterized. After optimization, about 1mg total of the major active compounds, burkholdines Bk-1229 (1) andBk-1097 (2), was obtained and structurally characterized from a 170 Lfermentation.⁶

Burkholdines represent a large family of cyclic lipopeptides isolatedfrom Burkholderia strains (especially B. ambifaria, B. contaminans, andB. cepacia).⁶ Occidiofungins, from B. contaminans, were recently shownto be nearly identical to burkholdines 1 and 2.⁷ Like burkholdines,occidiofungins are potent antifungal agents, which produce fungal cellmembrane defects.⁸ The occidiofungins could also clear Candidainfections in a mouse model.⁹ Between early work on B. ambifaria 2.2Nand this work on occidiofungins, these lipopeptides appear to bepromising antifungal agents.

Burkholderia strains are often potent pathogens of humans and otherorganisms.¹⁰ Many, but not all, strains of pathogenic Burkholderia,including B. cepacia (=Pseudomonas cepacia) secrete potent hemolytictoxins, which are chemically undefined lipopeptides.¹¹⁻¹⁵ Recently, onegroup of these toxins was shown to be required for full virulence.¹⁶

Occidiofungins are biosynthesized by a nonribosomal peptide synthetasemechanism.^(7,8) Analysis of other Burkholderia genome sequences inGenBank reveals that this gene cluster is widespread and probablyunderlies the biosynthesis of this compound family. Numerous mutationsof an occidiofungin-like gene cluster in B. cepacia abolished bothhemolysis and full virulence.¹⁶ Knockout of a glycosyltransferase thatlikely adds xylose to burkholdines/occidiofungins produced anintermediate stage of hemolysis and virulence, indicating that thisxylose may be important to toxicity. In addition, in the same paper theauthors report that 10 out of 54 B. cepacia complex isolates encoded anoccidiofungin-like gene cluster.¹⁶ One problem encountered in earlyoptimization of burkholdines was the emergence of anon-burkholdine-producing colonial variant at a frequency of 1-5% oftotal cells. Emergence of the non-burkholdine-producing colonial variantis apparently due to a large deletion, including loss of a ribosomaloperon (Cain and Falkinham, unpublished). This in part explains thevariation observed in the presence or absence of the occidiofunginbiosynthetic genes and could help to explain the prevalence of differentvirulence factors.

Here, material from several further fermentations was processed to yieldnew burkholdines 1215 (3), 1119 (4), 1213 (5). Their structures weredetermined using spectroscopic methods, and the absolute configurationof 1-5 was defined for the first time. The biological effects of 1-5were assessed using a panel of therapeutically important fungi, as wellas sheep erythrocytes. Although knockout of a glycosyltransferasereduced hemolysis in previous reports,¹⁶ presence or absence of xylosedid not correlate with hemolytic activity. Instead, compounds 3 and 4,in which 2,4-diaminobutyric acid (DAB) was present in place ofasparagine, were much more selective for fungi.

a. Results and Discussion

Burkholdine 1215 (3) was isolated as a pale yellow solid and assignedthe molecular formula C₅₂H₈₅N₁₁O₂₂ on the basis of HRESIMS (m/z1216.5960 [M+H]⁺⁾analysis. Analysis of the 2D NMR spectra includingCOSY, HSQC, and HMBC in DMSO-d₆ (Table 12 and FIG. 1) allowed for thecomplete spectral assignment of eight amino acids: one unit each ofglycine, Asn, 3-hydroxy-asparagine (3-OH-Asn), 3-hydroxy-tyrosine(3-OH-Tyr); DAB; two units of serine; and a fatty acyl amino acid (FAA).In comparison to the previously reported compound 1,⁶ 3 differed only bythe absence of an Asn; instead, a DAB residue was assigned to 3. Thexylose residue was assigned using COSY and ROESY correlations (FIG. 1).Large proton-proton coupling constants (J_(H2′-H3′)=8.7 Hz,J_(H3′-H4′)=9.0 Hz), a small coupling constant (˜2 Hz) between H-1′ andH-2′, and the chemical shift (99.3 ppm) of the anomeric carbon in xyloseestablished 3 as an α-xyloside. The placement of xylose on FAA C-7 wasestablished by an HMBC correlation between H-1′ and C-7 (FIG. 1) and aseries of ROESY correlations between H-1′ and H-6, H-7, H-8 (FIG. 1). Inthe related compound 1, the sugar is a β-xyloside. In comparison tof-xyloside 1, the ¹H NMR chemical shifts of H-5 and H-6 α-xyloside 3were down field by 0.98 and 0.74 ppm, respectively, while the chemicalshift of H-7 was upfield by 0.14 ppm. Similarly, the ³C chemical shiftsof C-5 (δ_(C)71.1) and C-6 (δ_(C)750.5) were 4.6 and 1.8 ppm downfield,respectively, while C-7 (δ_(C)750.4) was 1.0 ppm upfield.

The amino acid sequence of 3 was deduced from ROESY correlations betweena protons and neighboring residue NH groups (FIG. 1), with support fromHMBC correlations. The overall amino acid sequence was firmlyestablished, but based upon the NMR data the DAB and 3-OH-Tyr residuescould be linked via either the α- or γ-amine of DAB. For example, theROESY correlation between 2-NH_(2,4-Dab) and H-2_(3-OH-Tyr) isrelatively weaker than found in other amino acids in the series, and noconvincing HMBC data could define this bond. Therefore, a chemicalmethod was use that was recently applied to the analysis of a series ofunrelated cyclic peptides.¹⁷ Compound 3 was derivatized with1-fluoro-2,4-dinitrobenzene (FDNB), and the resulting compound washydrolyzed in acid to yield a single dinitrophenyl-(DNP)-DAB derivative.The hydrolysate was compared using HPLC with authentic standards of DABderivatized in either the α- or γ-position, leading to theidentification of the γ-derivatized DAB in the natural product.Therefore, the amide bond was unambiguously shown to occur via theα-amine.

It was previously reported that the absolute configuration of 2 is:(FAA)-L-threo-OH-Asn; L-Ser; L-Asn; Gly; D-Asn; (OH-Tyr); D-Ser.⁶ Theconfigurations of the 3-OH-Tyr and the FAA were not reported. Here,chemical methods were used to compare 2 with new compound 3, and thepreviously unknown residues were also assigned. The absoluteconfiguration of amino acyl α carbons was assigned using the advancedMarfey's method.^(18,19) Compounds 2 and 3 were hydrolyzed separately,and the resulting amino acids were converted to bothNα-(2,4-dinitro-5-fluorophenyl)-L-leucinamide (L-FDLA) andNα-(2,4-dinitro-5-fluorophenyl)-D-leucinamide (D-FDLA) derivatives,which were characterized by LC-MS in comparison to the retention timefor D- and L-FDLA derivatives (Supporting Information). Using the FDLAderivatives and in comparison to 2, L-threo-OH-Asn was identified. Inaddition, in comparison to authentic standards reported by Fuji et.al,¹⁸ both D- and L-Ser and L-Asx were identified in 2 and 3, whileD-Asx was identified only in 2. Therefore, amino acids of previouslydetermined configuration were identical in both 2 and 3. In addition, 3contained DAB, while 2 did not. Using an authentic L-2,4-DAB standard,it was confirmed that the retention time for the D-FDLA-D-2,4-DABderivative was shorter than the L-FDLA-D-2,4-DAB derivative. Thus,D-2,4-DAB was identified in 3.

In the previous description of 1 and 2, the configurations of FAA and3-OH-Tyr were not reported.⁶ FDLA-3-OH-Tyr was not detected which wasapparently destroyed by hydrolysis. To determine the relativeconfiguration of this residue in both compounds, the coupling constantwas examined between the alpha and beta protons in 3-OH-Tyr and itsderivatives. In literature reports, this value was was between 3.5 Hzand 4.8 Hz for the anti-configuration, while in the syn-configuration itwas between 4.6 Hz and 6.6 Hz (Supporting Information).²⁰⁻²² Since theobserved coupling constant between H-2_(3-OH-Tyr) and H-3_(3-OH-Tyr) wassmall (3.0 Hz), the relative configuration of 3-OH-Tyr in 2 was assignedas anti (2R*,3R*).

Fuji et. al applied the advanced Marfey's method to determine theabsolute configuration of primary amino compounds.²³ In this method, themore hydrophobic side of the molecule connected to CHNH₂ is assigned asR₁ on the left, while the more hydrophilic side is assigned as R₂ andplaced on the right. If the D-FDLA derivative elutes earlier than theL-FDLA derivative, then the amine group would be drawn backward out ofthe plane in relationship to R₁ and R₂. Indeed, in this case the D-FDLAderivatives of FAAs from both 2 and 3 eluted earlier than the L-FDLA,indicating that these amino acids should be assigned as R (SupportingInformation). A weakness of this method is that it has only been appliedto a few substrates.

In order to determine the relative configuration of FAA in 2, a1D-TOCSY-MDEC²⁴ experiment was applied to obtain the ¹H-¹H couplingconstants (H-3/H-4a: 1.0 Hz; H-3/H-4b: 11.0 Hz; H-5/H-4a: 11.0 Hz;H-5/H-4b: 1.0 Hz, H-5/H-6: 6.7 Hz; H-6/H-7: 1.0 Hz) for the FAA moietyof 2. Because of the large couplings between H-3/H-4b and H-5/H-4a, anda series of ROESY correlations (H-3/H-5; H-3/5-OH; H-2/H-4a,b;H-6/H-4a,b; H-3/H-5; H-3/5-OH), the absolute configuration of C-5 wasassigned as R. Likewise, based on the ¹H-¹H couplings and ROESYcorrelations, an anti form relative configuration was assigned for bothC-5-C-6 and C-6-C-7 bonds (FIG. 2). Thus, the absolute configuration ofFAA was determined as 3R,5R,6S,7S. Lastly, in order to determine theabsolute configuration of the carbohydrate unit in burkholdines, 1 washydrolyzed in 1N HCl and per-acetylated. D-xylose was identified bycomparing its retention time by GC-MS to authentic standards ofacetylated D- and L-xylose.

Burkholdine 1119 (4) was isolated as a pale yellow solid. Its molecularformula was determined as C₅₂H₈₅N₁₁O₂₁ on the basis of HRESIMS (m/z1200.6010 [M+H]⁺) data, which differed from 3 by the loss of an oxygenatom. Analysis of the 2D NMR spectra including COSY and HSQC in DMSO-d₆(Table 12) revealed that 2 had one unit each of Gly, 3-OH-Tyr, DAB, andFAA, and two units of Ser and Asn. Compound 4 differed from 3 only bythe absence of a 3-OH-Asn; instead, one more Asn was assigned to 4. Thechemical shifts of the oxygenated methines in FAA of 4 were very similarto those of 1 (Table 12), In addition, the anomeric carbon in xylose of4 showed a larger chemical shift (102.7 ppm). These data indicated that4 was a f-xyloside. The absolute configurations of the amino acidresidues were confirmed to be the same as those in 3, using the advancedMarfey's method.^(18,19)

Burkholdine 1213 (5) was isolated as a pale yellow solid. Its molecularformula was determined as C₅₂H₈₃N₁₁O₂₂ on the basis of HRESIMS (m/z1214.5850 [M+H]⁺) data, which differed from 4 by 14 Da. The NMR data of5 were largely identical to those of 4. In fact, the only majordifference was that the DAB of 5 was replaced with Asn, as is found in 1and 2. Complete analysis of 1D and 2D NMR data (Supporting Information)showed that the other seven amino acids were identical in both 4 and 5.Additionally, ROESY confirmed the sequence to be identical (FIG. 1). Theabsolute configurations of the amino acid residues in 5 were examined bythe advanced Marfey's method^(18,19) and confirmed to be identical tothose in other members of this series.

In addition to the reported metabolites 1-5, several other minor orinseparable components were present in the mixture and remain to bedescribed.

TABLE 12 3 unit No δ_(C) δ_(H) (J in Hz) 3-OH-Tyr  1 170.7, C —  2 58.9,CH 4.25, dd (11.0, 3.0) 2-NH — 8.21, d (11.0)  3 71.7, CH 5.07, brs 3-OH— 5.69, brs  4 131.0, C — 5, 6 127.5, CH 7.07, d (8.5) 8, 9 115.3, CH6.65, d (8.5)  7 157.3, C — 2,4-Dab  1 172.1, C —  2 51.9, CH 4.04, m2-NH — 6.06, brs  3 30.4, CH₂ 1.75, m, 2.34, m  4 37.4, CH₂ 3.05, m,2.59, m Gly  1 170.9, C —  2 44.0, CH₂ 3.77, m, 3.63, m 2-NH 9.74, brsAsn  1 173.0, C —  2 49.6, CH 4.74, dd (8.0, 4.0) 2-NH 8.93, d (7.6)  32.77, dd (8.0, 16.0), 2.24, dd (16.0, 4.0)  4 172.2, C — 4-NH₂ — 7.14,s, 6.67, s Ser I  1 169.6, C —  2 57.6, CH 3.96, t (6.0) 2-NH 8.07, m  360.2, CH₂ 4.43, m, 3.32, m 3-OH-Asn  1 172.7, C —  2 61.0, CH 4.45, m2-NH — 6.80 brs  3 73.2, CH 3.90, d (4.5)  4 169.6, C — 4-NH₂ — 7.39, s,6.80, s FAA  1 170.9, C —  2 41.0, CH₂ 2.72, m, 2.53, m  3 45.0, CH4.20, m 3-NH — 9.82, brs  4 33.1, CH₂ 1.89, m, 1.53, m  5 71.1, CH 4.46m  6 75.5, CH 3.80 m  7 75.4, CH 3.58 m  8 29.4, CH₂ 1.51 m, 1.43 m 9-1723-32, CH₂ 1.24 m 18 14.8, CH₃ 0.82 t (6.5) Ser II  1 171.6, C —  254.8, CH 4.22 m 2-NH — 8.33 d (6.6)  3 59.1, CH₂ 3.45 m 3-OH

Table 13. Antifungal and hemolytic activity of burkholdines (note:antifungal values for 1 and 2 previously reported⁶). Antifungal activityis reported as an average of triplicate measurement. Hemolytic activityis concentration required for 10% hemolysis, with values indicatingmean±standard deviation from 2 independent measurements. Thehemolytic:antifungal (H:F) ratio is approximate and reported usingvalues for the least-potent antifungal activity (higher ratio=moreselective antifungal). NT: not tested.

TABLE 13 Minimal Inhibitory Concentration 10% (μg/mL) Hemolysis RatioCompound S. cerevisiae C. albicans A. niger (μg/mL) H:F 1 0.4 12.5 12.5 10 ± 2.8 1 2 1.6 12.5 1.6 4.5 ± 3.5 0.4 3 0.15 0.15 0.15   5 ± 3.9 33 40.1 0.4 0.1  15 ± 7.1 38 5 2.0 31.0 2.0 37 ± 12 1 Amphotericin 2.5 2.52.5 NT NA B Triton X NT NT NT  48 ± 3.5 NA

The biological activities of burkholdines were examined against a panelof fungi (Table 13). Bk-1119 (4) exhibited the greatest potency, with25-fold greater activity than the clinically used agent, amphotericin B,against the filamentous fungus, Aspergillus niger. In addition, thecompounds were measured for lysis of sheep erythrocytes. Compoundscontaining Asn adjacent to 3-OH-Tyr (1, 2, and 5) exhibited roughlyequal antifungal and hemolytic potencies, although this wasfungal-strain dependent. For example, 1 was selectively active againstSaccharomyces cerevisiae in comparison to hemolysis, but exhibited noselectivity for A. niger or Candida albicans. By contrast, compoundscontaining DAB adjacent to 3-OH-Tyr (3 and 4) were highly selective forfungi versus hemolysis, with 4 exhibiting both the highest potency andhighest selectivity in the series. This activity compares very favorablywith that of amphotericin B, which was less active than 4 and is wellknown for its narrow therapeutic index.

The burkholdines have been in continuous development as antifungalagents since 1996 because of the potent biological activity of theproducing strain,²⁵⁻²⁷ although the burkholdines structures remainedunknown until recently. Indeed, difficulties in purification greatlydelayed an understanding of this compound family. Many lipopeptidesrelated to burkholdines were previously isolated from the Burkholderiagroup, but the lack of pure material greatly complicated structuredetermination.^(7,8, 28,29) Based upon careful analysis and comparisonof literature data for these compounds with the burkholdines datapresented here, it is proposed that these compounds are actually allhighly related, or possibly even identical, to the burkholdines class ofcompounds (Supporting Information). Further work is required to supportor refute this hypothesis. The implication is that, where hemolyticactivity is important for Burkholderia virulence,¹⁶ mounting evidenceindicates that these peptides are the likely virulence factors.

Here, using the purified compounds, it was shown that some of theburkholdines are exceptionally potent antifungal agents. In particular,aspergilli are important targets, representing invasive fungi that leadto high mortality rates, and for which current therapies are extremelylimited.¹ Therefore, the exceptional potency of these compounds ispromising in the development of new agents to solve serious problems inhuman health. Hemolysis and the resulting toxicity would obviouslyprevent the widespread application of these compounds as antifungalagents. The structure-activity data presented here reveal thatselectivity is possible, and in fact at least some of the compounds aremore selective and more potent than the clinically applied amphotericinB, which is a classic narrow-therapeutic index drug. In one study,amphotericin B provoked 10% hemolysis at ˜2 μg/mL, for example.³⁰ Thesecompounds are thus important for their potential both in understandingand in treating human infective diseases.

b. Experimental Methods and Procedures

(1) General Experimental Procedures

UV spectra were obtained using a Perkin-Elmer Lambda2 UV/visspectrometer. NMR data were collected using either a Varian INOVA 500(¹H 500 MHz, ¹³C 125 MHz) NMR spectrometer with a 3 mm Nalorac MDBGprobe or a Varian INOVA 600 (¹H 600 MHz, ¹³C 150 MHz) NMR spectrometerequipped with a 5 mm ¹H[¹³C,¹⁵N] triple resonance cold probe with az-axis gradient, utilizing residual solvent signals for referencing.High-resolution mass spectra (HRMS) were obtained using a Bruker(Billerica, Mass.) APEXII FTICR mass spectrometer equipped with anactively shielded 9.4 T superconducting magnet (Magnex Scientific Ltd.),an external Bruker APOLLO ESI source, and a Synrad 50W CO₂ CW laser.Supelco Discover HS (4.6×150 mm) and semipreparative (10×150 mm) C₁₈ (5μm) columns were used for HPLC on a Hitachi Elite Lachrom Systemequipped with a diode array L-2455 detector.

(2) Fermentation and Extraction

Cultures (170 L) of strain 2.2N in a medium consisting 1.5% (w/v)Tryptic Soy Broth, 0.2% (w/v) sucrose, 0.25% (w/v) K₂HPO₄, and 0.5%(w/v) NH₄Cl were prepared and incubated for 32 h at 30° C. with aerationmaintained at 30% dissolved oxygen. pH 6 was maintained by addition of17% (v/v) NH₄OH. Cells were recovered by centrifugation (5000×g) in acontinuous flow centrifuge and cell pastes were spray dried at an inlettemperature of 170° C. and outlet temperature of 100° C. A slurry ofspray dried cells (100 g) in dichloromethane (DCM, 300 mL) was stirredat room temperature for 2 h. The slurry was centrifuged and the solidresidue was stirred with 2-propanol (300 mL) for 1 h. The solvent wasremoved by centrifugation. The supernatant was discarded and the residuewas stirred with CH₃CN:H₂O (1:1; 200 mL) for 4 h at room temperature.The resulting suspension was centrifuged to clarity. The supernatant wasdried under blowing air for 1 h, providing extract A. The remainingpellet was stirred in DMSO (200 mL) for 1 hour and centrifuged toclarity. The supernatant was dried under vacuum and suspended inCH₃CN:H₂O (1:1; 50 mL) followed centrifugation to clarity. Thesupernatant was dried under blowing air overnight, providing extract B.Extracts A and B were combined to yield a crude mixture (1.1 g) enrichedin burkholdines.

(3) Purification.

The combined extract (1.1 g) was dissolved in CH₃CN:H₂O (1:1; 100 mL)and added to HP20SS resin (60 g). The solvent was removed in vacuo, andthe dried resin was extracted using a gradient of H₂O to CH₃CN in 25%steps. The burkholdines were found in 50%, 75%, and 100% CH₃CN fractionsusing an antifungal assay. The burkholdines were next enriched bySephadex LH-20 in CH₃CN:H₂O (1:1). Fractions containing burkholdineswere further purified by C₁₈ HPLC using 35% CH₃CN in H₂O with 0.1% TFAto obtain fraction I (1.1 mg, t_(R)=10.9 min) containing twounidentified Bks with molecular weights of 1361 and 1377, respectively;fraction II containing 3 and 4 (2.1 mg, t_(R)=13.8 min), 1 (3.0 mg,t_(R)=15.7 min), and 5 (400 μg, t_(R)=16.2 min); fraction III (100 μg,t_(R)=24.0 min) containing two unidentified Bks with molecular weightsof 1083 and 1067, respectively; and fraction IV (5.0 mg, t_(R)=30.0 min)containing 2 and an unidentified Bk with molecular weights of 1081.Fraction II was further purified by C₁₈ HPLC using 30% CH₃CN in H₂O with0.1% TFA to obtain 3 (700 μg, t_(R)=36.8 min) and 4 (200 μg, t_(R)=38.3min).

(4) BK-1215 (3)

Bk-1215 (3) is a pale yellow solid; UV (CH₃CN:H₂O 1:1) λ_(max) (log ε)230, 274 nm; ¹H and ¹³C NMR (see Table 12), ¹H NMR (DMSO-d₆, 600 MHz)for xylose unit δ 4.26 (1H, m, H-1′), 3.17 (1H, brd, J=8.7 Hz, H-2′),3.08 (1H, dd, J=9.0, 8.7 Hz, H-3′), 3.29 (1H, m, H-4′), 3.67 (1H, m,H-5′a), 2.99 (1H, m, H-5′b). ¹³C NMR (DMSO-d₆, 150 MHz) δ 99.3 (CH,C-1′), 73.9 (CH, C-2′), 77.5 (CH, C-3′), 70.4 (CH, C-4′), 66.6 (CH₂,C-5′); HRESIMS m/z 1216.5960 [M+H]⁺ (calcd for C₅₂H₈₅N₁₁O₂₂, 1216.5949,0.9 ppm).

(5) BK-1119 (4)

Bk-1119 (4) is a pale yellow solid; UV (CH₃CN:H₂O 1:1) λ_(max) (log ç)230, 274 nm; ¹H NMR (DMSO-d₆, 600 MHz) δ 4.13 (1H, m, H-2_(3-OH-Tyr)),8.05 (1H, m, 2-NH_(3-OH-Tyr)), 5.03 (1H, brs, H-3_(3-OH-Tyr)), 5.68 (1H,s, 3-OH_(3-OH-Tyr)), 7.12 (2H, d, J=8.0 Hz, H-5,6_(3-OH-Tyr)), 6.63 (2H,d, J=8.0 Hz, H-8,9_(3-OH-Tyr)), 4.35 (1H, m, H-2_(2,4-Dab)), 7.69 (1H,m, 2-NH_(2,4-Dab)), 2.03 (1H, m, H-3a_(2,4-Dab)), 1.86 (1H, m,H-3b_(2,4-Dab)), 2.86 (2H, m, H-4_(2,4-Dab)), 3.72 (1H, m, H-2a_(Gly)),3.63 (1H, m, H-2b_(Gly)), 7.95 (1H, brs, 2-NH_(Gly)), 4.52 (1H, m,H-2_(Asn I)), 8.29 (1H, m, 2-NH_(Asn I)), 2.37 (1H, m, H-3a_(Asn I)),2.56 (1H, m, H-3b_(Asn I)), 4.18 (1H, m, H-2_(SerI)), 8.02 (1H, m,2-NH_(SerI)), 3.38 (1H, m, H-3a_(Ser I)), 3.29 (1H, m, H-3b_(Ser I)),4.48 (1H, m, H-2_(Asn II)), 8.04 (1H, m, 2-NH_(Asn II)), 2.48 (1H, m,H-3a_(Asn II)), 2.31 (1H, m, H-3b_(Asn II)), 2.36 (1H, m, H-2a_(FAA)),2.29 (1H, m, H-2b_(FAA)), 4.11 (1H, m, H-3_(FAA)), 7.48 (1H, brs,2-NH_(FAA)), 1.72 (1H, m, H-4a_(FAA)), 1.40 (1H, m, H-4b_(FAA)), 3.40(1H, m, H-5_(FAA)), 3.07 (1H, m, H-6_(FAA)), 3.68 (1H, m, H-7_(FAA)),1.51 (1H, m, H-8a_(FAA)), 1.50 (1H, m, H-8b_(FAA)), 1.24 (18H, m,H-9-17_(FAA)), 0.82 (3H, t, J=6.5 Hz, H-18_(FAA)). 4.14 (1H, m, H-1′),2.94 (1H, m, H-2′), 3.06 (1H, m, H-3′), 3.26 (1H, m, H-4′), 3.67 (1H, m,H-5′a), 3.01 (1H, m, H-5′b), 4.16 (1H, m, H-2_(SerII)), 7.72 (1H, m,2-NH_(Ser II)), 3.55 (2H, m, H-3_(Ser II)), 4.78 (1H, brs,3-OH_(Ser II)); ¹³C NMR (DMSO-d₆, 150 MHz) δ 60.8 (CH, C-2_(3-OH-Tyr)),71.7 (CH, C-3_(3-OH-Tyr)), 128.0 (CH, C-5,6_(3-OH-Tyr)), 115.4 (CH,C-8,9_(3-OH-Tyr)), 51.3 (CH, C-2_(2,4-Dab)), 30.3 (CH₂, C-3_(2,4-Dab)),36.7 (CH₂, C-4_(2,4-Dab)), 42.8 (CH₂, C-2Gly), 50.4 (CH, C-2_(Asn I)),37.5 (CH₂, C-3_(Asn I)), 56.4 (CH, C-2_(SerI)), 61.9 (CH₂, C-3_(SerI)),50.5 (CH, C-2_(Asn II)), 37.9 (CH, C-3_(Asn II)), 41.4 (CH₂, C-2_(FAA)),45.1 (CH, C-3_(FAA)), (C-4_(FAA), not detected), 67.9 (CH, C-5_(FAA)),75.6 (CH, C-6_(FAA)), 77.8 (CH, C-7_(FAA)), 30.6 (CH₂, C-8_(FAA)), 23-32(CH₂, C-9-17_(FAA)), 14.8 (CH₃, C-18_(FAA)), 102.7 (CH, C-1′), 73.7 (CH,C-2′), 77.2 (CH, C-3′), 70.2 (CH, C-4′), 66.3 (CH₂, C-5′), 56.3 (CH,C-2_(Ser II)), 61.9 (CH₂, C-3_(Ser I)); HRESIMS m/z 1200.6010 [M+H]⁺(calcd for C₅₂H₈₅N₁₁O₂₁, 1200.6000, 0.9 ppm).

(6) BK-1213 (5)

Bk-1213 (5) is a pale yellow solid; UV (CH₃CN:H₂O 1:1) λ_(max) (log ε)230, 274 nm; ¹H NMR (DMSO-d₆, 600 MHz) δ 4.10 (1H, m, H-2_(3-OH-Tyr)),8.04 (1H, d, J=8.0 Hz, 2-NH_(3-OH-Tyr)), 4.97 (1H, m, H-3_(3-OH-Tyr)),5.65 (1H, s, 3-OH_(3-OH-Tyr)), 7.15 (2H, d, J=8.5 Hz, H-5,6_(3-OH-Tyr)),6.66 (2H, d, J=8.5 Hz, H-^(8,9) _(3-OH-Tyr)), 4.52 (1H, m, H-2_(Asn I)),7.89 (1H, m, 2-NH_(Asn I)), 2.60 (1H, m, H-3a_(Asn I)), 2.45 (1H, m,H-3b_(Asn I)), 3.57 (1H, m, H-2a_(Gly)), 3.77 (1H, m, H-2b_(Gly)), 7.83(1H, brs, 2-NH_(Gly)), 4.46 (1H, m, H-2_(Asn, I)), 8.27 (1H, d, J=6.0Hz, 2-NH_(Asn II)), 2.64 (1H, m, H-3a_(Asn II)), 2.39 (1H, m,H-3b_(Asn II)), 4.23 (1H, m, H-2_(SerI)), 7.74 (1H, d, J=6.8 Hz,2-NH_(SerI)), 3.45 (1H, m, H-3a_(SerI)), 3.37 (1H, m, H-3b_(SerI)), 4.52(1H, m, H-2_(AsnII)), 7.97 (1H, brs, 2-NH_(AsnII)), 2.52 (1H, m,H-3a_(Asn II)), 2.33 (1H, m, H-3b_(Asn II)), 2.99 (1H, m, H-²a_(FAA)),2.37 (1H, m, H-2b_(FAA)), 4.18 (1H, m, H-3_(FAA)), 7.50 (1H, brs,2-NH_(FAA)), 1.74 (1H, m, H-4a_(FAA)), 1.40 (1H, m, H-4b_(FAA)), 3.44(1H, m, H-5_(FAA)), 3.08 (1H, m, H-6_(FAA)), 3.70 (1H, m, H-7_(FAA)),1.54 (2H, m, H-8_(FAA)), 1.24 (18H, m, H-9-17_(FAA)), 0.82 (3H, t, J=6.5Hz, H-18_(FAA)). 4.18 (1H, m, H-1′), 2.99 (1H, m, H-2′), 3.09 (1H, m,H-3′), 3.32 (1H, m, H-4′), 3.70 (1H, m, H-5′a), 3.05 (1H, m, H-5′b),4.18 (1H, m, H-2_(SerII)), 8.01 (1H, m, 2-NH_(Ser I)), 3.59 (2H, m,H-3_(Ser II)); ¹³C NMR (DMSO-d₆, 150 MHz) δ 61.4 (CH, C-2_(3-OH-Tyr)),71.7 (CH, C-3_(3-OH-Tyr)), 128.0 (CH, C-5,6_(3-OH-Tyr)), 115.2 (CH,C-8,9_(3-OH-Tyr)), 50.7 (CH, C-2_(Asn I)), 37.9 (CH₂, C-3_(Asn I)), 43.1(CH₂, C-2_(Gly)), 50.9 (CH, C-2_(Asn II)), 37.6 (CH₂, C-3_(Asn II)),56.1 (CH, C-2_(Ser I)), 62.3 (CH₂, C-3_(Ser I)), 50.7 (CH,C-2_(Asn III)), 38.2 (CH, C-3_(Asn III)), 41.8 (CH₂, C-2_(FAA)), 45.1(CH, C-3_(FAA)), 33.9 (CH₂, C-4_(FAA)), 68.2 (CH, C-5_(FAA)), 75.2 (CH,C-6_(FAA)), 780.1 (CH, C-7_(FAA)), 30.7 (CH₂, C-8_(FAA)), 23-32 (CH₂,C-9-17_(FAA)), 14.8 (CH₃, C-18_(FAA)), 102.8 (CH, C-1′), 73.4 (CH,C-2′), 77.1 (CH, C-3′), 70.1 (CH, C-4′), 66.4 (CH₂, C-5′), 56.3 (CH,C-2_(Ser I)), 61.9 (CH₂, C-3_(Ser I)); HRESIMS m/z 1214.5850 [M+H]⁺(calcd for C₅₂H₈₃N₁₁O₂₂, 1214.5792, 4.7 ppm).

(7) Acid Hydrolysis of Peptides

Compounds 1-5 (30 μg each) were separately dissolved in 6 N HCl (500 μL)and heated in sealed ampule vials at 110° C. for 16 h. the solvent wasremoved in vacuo.

(8) LC-Ms Analysis of D/L-FDLA Derivatives

The acid hydrolysates were dissolved in H₂O separately. To a 50 μLaliquot of each were added 1 N NaHCO₃ (20 μL) and1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (1% solution in acetone, 100μL), and the mixture was heated to 40° C. for 50 min. The solutions werecooled to room temperature, neutralized with 1 N HCl (20 μL), and thendried in vacuo. The residues were dissolved in 1:1 CH₃CN—H₂O and thenanalyzed by LC-MS. The analysis of the L- and D-FDLA derivatives wasperformed on a Supelcosil LC-18 column (150×4.6 mm, 5 μm) employing alinear gradient of from 25% to 80% CH₃CN in 0.01 M formic acid at 0.5mL/min over 55 min. The retention time of the D- and L-FDLA derivativeswere as follows: D-2,4-DAB: 38.53, 39.21 min, m/z 705 [M−H]⁻; D-Ser:21.15, 20.39 min, m/z 398 [M−H]⁻; L-3-OH-Asn: 18.16, 18.86 min, m/z 442[M−H]⁻; D-FAA: 49.35, 52.03 min, m/z 640 [M−H]⁻; L-Asn: 21.61, 20.82min, m/z 426 [M−H]⁻.

(9) Absolute Configuration of Xylose

Compound 1 (0.1 mg) was dissolved in 1NHCl (250 μL) and heated at 80° C.for 4 h with stirring. After cooling, the solvent was removed in vacuoand the residue was dissolved in pyridine anhydrous (200 L) and aceticanhydride (10 μL). The solution was stirred at room temperature for 16h. The product was analyzed by GC-MS. A 30 m×0.25 mm i.d. RestekRT-bDEXm column was used for enantioselective GC with the startingtemperature at 75° C. and final temperature at 230° C. at a rate of 1°C./min. A Waters GCT Premier time-of-flight mass spectrometer wasemployed for analysis. The retention time of the acetyl derivatives ofthe D and L-xylose standards was 68.68 and 69.02 min, respectively. Theacetyl derivative of D-xylose in the hydrolysate of 1 had retentiontimes of 68.65 min.

(10) Preparation of Alpha and Gamma-DNP-L-2,4-Dab

L-2,4-DAB (11.0 mg) and sodium carbonate (anhydrous, 20.0 mg) weredissolved in water (10 mL) at 40° C. FDNB (18.6 mg) was added, and themixture was stirred vigorously by means of a magnetic stirrer at 40° C.The finely divided suspension of FDNB disappeared after about 30 min,indicating that the reaction was complete. Acidification of theresulting solution with concentrated HCl yielded mixture of α- andγ-DNP-L-2,4-DAB, which were further purified using HPLC C₁₈ to yieldpure α- and γ-DNP-L-2,4-DAB, respectively.

Antifungal activity was measured using the previously described method.⁶

(11) Measurement of Hemolysis

Hemolytic activity of the compounds was measured against a 0.25% sheepred blood cell suspension in phosphate buffered saline. Compounds wereadded to the red blood cell suspension in two-fold serial dilutions andafter 1 h incubation at 37° C., the red blood cells were pelleted(1,000×g for 5 min), supernatant (100 μL) was transferred to wells of a96-well, flat-bottom plate, and the absorbance at 540 nm was measured.Results are reported as concentration of compound resulting in 10%hemolysis using Triton X-100 as the positive, complete lysis control.

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The invention claimed is:
 1. A pharmaceutical composition comprising acompound having a structure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is α-xylose or β-xylose;and wherein R³ and R⁴ are each hydrogen; or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrierselected from aerosol, cream, ointment, lotion, dusting powder,liposome, microcapsule, emulsion, and polymeric encapsulation orcombination thereof.
 2. The composition of claim 1, wherein R¹ ishydrogen.
 3. The composition of claim 1, wherein R¹ is hydroxyl.
 4. Thecomposition of claim 1, wherein R² has a structure represented by aformula:


5. The composition of claim 1, wherein R² has a structure represented bya formula:


6. The composition of claim 1, wherein R¹ is hydroxyl; R² is α-xylose;and R³ and R⁴ are each hydrogen.
 7. The composition of claim 1, whereinR¹ is hydrogen; R² is β-xylose; and R³ and R⁴ are each hydrogen.
 8. Thecomposition of claim 1, wherein the compound is cytotoxic.
 9. Thecomposition of claim 1, wherein the compound exhibits antifungalactivity against Alternaria, Aspergillus, Botrytis, Cercospora,Cercosporidium, Erysiphe, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Septoria, Sporothrix, Stemphylium,Trichophyton, or Verticillium.
 10. The composition of claim 1, whereinthe compound is isolated from a culture of Burkholderia ambifaria.
 11. Amethod for the treatment of an animal having a fungal infection,comprising administering to the animal a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an effective amountof a compound, or a pharmaceutically acceptable salt thereof, having astructure represented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is α-xylose or β-xylose;and wherein R³ and R⁴ are each hydrogen, thereby treating the fungalinfection in the animal, wherein the fungal infection is selected frominfections of Botrytis sp., Mycosphaerella sp., Cercospora sp.,Rhizoctonia sp., Sclerotinia sp., Pythium sp., Phytophthora sp.,Alternaria sp., Saccharamyces sp., Candida sp., Aspergillus sp.,Pseudocercosporella sp., Cladosporium sp., Penicillium sp., Chaetomiumsp., Fusarium sp., Pyricularia sp., Erysiphe sp., Spaerotheca sp.,Leptosphaeria sp, Plasmopara sp., and Colletotrichum sp.
 12. The methodof claim 11, wherein the fungal infection is cutaneous.
 13. A method forthe treatment of a plant having a fungal infection, comprisingadministering to the plant an agricultural composition comprising aagriculturally acceptable carrier and an effective amount of a compound,or a pharmaceutically acceptable salt thereof, having a structurerepresented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R¹ is hydrogen or hydroxyl;wherein R² is α-xylose or β-xylose; and wherein R³ and R⁴ are eachhydrogen, thereby treating the fungal infection in the plant, whereinthe fungal infection is selected from infections of Botrytis sp.,Mycosphaerella sp., Cercospora sp., Rhizoctonia sp., Sclerotinia sp.,Pythium sp., Phytophthora sp., Alternaria sp., Saccharamyces sp.,Candida sp., Aspergillus sp., Pseudocercosporella sp., Cladosporium sp.,Penicillium sp., Chaetomium sp., Fusarium sp., Pyricularia sp., Erysiphesp., Spaerotheca sp., Leptosphaeria sp, Plasmopara sp., andColletotrichum sp.
 14. The method of claim 13, wherein the applicationis foliar.
 15. A pharmaceutical composition comprising apharmaceutically acceptable salt of a compound having a structurerepresented by a formula:

wherein R¹ is hydrogen or hydroxyl; wherein R² is α-xylose or β-xylose;and wherein R³ and R⁴ are each hydrogen, and a pharmaceuticallyacceptable carrier.
 16. The composition of claim 15, wherein R¹ ishydrogen.
 17. The composition of claim 15, wherein R¹ is hydroxyl. 18.The composition of claim 15, wherein R² has a structure represented by aformula:


19. The composition of claim 15, wherein R² has a structure representedby a formula:


20. The composition of claim 15, wherein R¹ is hydroxyl; R² is α-xylose;and R³ and R⁴ are each hydrogen.
 21. The composition of claim 15,wherein R¹ is hydrogen; R² is β-xylose; and R³ and R⁴ are each hydrogen.22. The composition of claim 15, wherein the compound is cytotoxic. 23.The composition of claim 15, wherein the compound exhibits antifungalactivity against Alternaria, Aspergillus, Botrytis, Cercospora,Cercosporidium, Erysiphe, Geotrichum, Mycosphaerella, Mucor,Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia,Pythium, Rhizoctonia, Rhizopus, Septoria, Sporothrix, Stemphylium,Trichophyton, or Verticillium.
 24. The composition of claim 15, whereinthe compound is isolated from a culture of Burkholderia ambifaria. 25.The composition of claim 15, wherein the pharmaceutically acceptablecarrier is selected from aerosol, cream, ointment, lotion, dustingpowder, liposome, microcapsule, emulsion, and polymeric encapsulation orcombination thereof.